Stella Ranuncolo - Academia.edu (original) (raw)

Papers by Stella Ranuncolo

Blood, Nov 16, 2006

The BCL6 transcriptional repressor is the most commonly involved oncogene in diffuse large B-cell... more The BCL6 transcriptional repressor is the most commonly involved oncogene in diffuse large B-cell lymphomas (DLBCL). Constitutive expression of BCL6 has been proposed to mediate lymphomagenesis through several mechanisms, including evasion of cell death, proliferation and differentiation blockade. We show here that BCL6 mediates these effects through distinct mechanisms. First, we show that blocking the association of the SMRT corepressor with BCL6 using our specific peptide inhibitor (BPI) abrogates only the survival effects of BCL6 but has no effect on differentiation. Accordingly, BPI upregulates survival genes such as ATR and p53, but not genes associated with differentiation such as Blimp1, XBP and Syndecan. In contrast, BCL6 shRNA upregulates both survival and differentiation genes and induces both cell death and differentiation. We and others have shown that BCL6 can also directly bind to the MTA3 corepressor, which is implicated in differentiation of Burkitt lymphoma cells. We found that BCL6 and MTA3 are co-expressed in DLBCL cells and primary human centroblasts (the precursor cell for most DLBCLs). The endogenous BCL6 and MTA3 proteins interacted in DLBCLs cells in co-immunoprecipitation experiments. In contrast to SMRT blockade with BPI, siRNA depletion of MTA3 induced expression of the Blimp1, XBP and Syndecan but not p53 and ATR. MTA3 depletion induced plasmacytic differentiation within 72 hours as shown in functional assays and by surface markers. We performed ChIP on chip using custom arrays densely tiling with oligonucleotides covering the entire genomic loci of 20 BCL6 target genes. Interestingly, BCL6 formed different types of repression complexes at differentiation genes (Complex with MTA3/NuRD) vs. survival genes (complex with SMRT and N-CoR). BCL6-mediated repression of genes involved in survival and differentiation thus depend on distinct biochemical mechanisms. The relevance of these findings for human disease was underscored by the fact that we found a statistically significant positive correlation between MTA3 and BCL6 gene expression in a database of 176 human DLBCLs…

Cancer Research, Apr 15, 2006

Proc Amer Assoc Cancer Res, Volume 47, 2006 5651 The BCL6 (B-Cell-Lymphoma-6) transcriptional rep... more Proc Amer Assoc Cancer Res, Volume 47, 2006 5651 The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas and is required for establishment of germinal centers by normal B-cells. However, the mechanisms by which BCL6 licenses germinal center formation and lymphomagenesis are unknown. To characterize this mechanism we identified BCL6 target genes by expression arrays and high throughput chromatin immunoprecipitations. Remarkably, a number of these target genes were critical mediators of DNA damage sensing checkpoints including ATR and p53. Therefore, we hypothesized that BCL6 could attenuate DNA damage sensing by silencing these genes, which is likely a critical attribute for survival and proliferation of germinal center B-cells undergoing somatic hypermutation (SHM) and class switch recombination (CSR). Accordingly, we found that expression of BCL6 in normal diploid fibroblasts could block cellular sensing of DNA damage as demonstrated by loss of histone 2AX (H2AX) phosphorylation and delayed repair of double strand breaks, shown by COMET assays. Repression of ATR (but not p53 or other targets) was required for this phenotype. This is a physiological effect since the same result was observed when BCL6 was expressed in purified primary human tonsilar naive B-cells. Reciprocally, shRNA knockdown of BCL6 in B-cell lymphomas rescued repression of ATR, enhanced H2AX phosphorylation and accelerated repair of double strand breaks, independent of the status of p53. shRNA knockdown of BCL6 caused a marked increase of apoptosis in lymphoma cells in response to DNA damage, due to restored DNA damage checkpoint functions. Importantly, BCL6 knockdown had an identical effect on ATR levels, H2AX phosphorylation, DNA damage, and survival in purified primary human germinal center centroblasts. These results suggest that a major role of BCL6 in germinal center formation is to attenuate cellular response to DNA damage occurring as a byproduct of CSR and SHM. The same mechanism also seems to be required for lymphomagenesis, since we observed that sustained BCL6 expression in human primary mature B-cells leads to aberrant survival properties and genomic instability. Moreover, BCL6 blockade using a specific inhibitor molecule designed by our lab induces apoptosis in lymphoma cells and synergizes with DNA damaging agents. Therefore, we have identified a critical mechanism of action of the BCL6 oncoprotein in normal and pathogenic states and show that specific targeting of BCL6 could synergize with chemotherapy drugs for the therapy of B-cell lymphomas.

Blood, Nov 16, 2008

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease in which subsets of tumors likel... more Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease in which subsets of tumors likely rely upon different survival pathways. We previously identified a subset of primary DLBCLs with increased abundance of multiple components of the BCR signaling cascade including the spleen tyrosine kinase, SYK (“BCR-type” tumors). In related studies, we found that BCR-type DLBCL cell lines and primary tumors exhibited tonic B-cell receptor signaling. Since BCR-associated SYK activation initiates downstream events and amplifies the original BCR signal, we previously evaluated the efficacy of targeted SYK inhibition. Inhibition of SYK-dependent tonic BCR signaling induced apoptosis in the majority of “BCR-type” cell lines and primary DLBCLs. Furthermore, an oral SYK inhibitor (FosD) had promising activity in a subset of relapsed/refractory DLBCLs in a recently completed phase I/II clinical trial. “BCR-type” tumors also have more abundant expression of the transcriptional repressor, BCL6, and more frequent BCL6 translocations, greater repression of BCL6 target genes and increased sensitivity to BCL6 inhibitors. Since the same subset of primary DLBCLs relies upon SYK-dependent BCR signaling and BCL6-mediated transcriptional repression, we evaluated potential connections between BCL6 and SYK. SYK is a major substrate of the tissue-specific and developmentally regulated protein tyrosine phosphatase, PTPROt. For this reason, we first assessed the relative expression of BCL6 and PTPROt in two large independent series of transcriptionally profiled primary DLBCLs and two independent groups of highly purified normal B-cell subpopulations (naïve, germinal center, memory). There was a highly significant reciprocal pattern of expression of PTPROt and BCL6 in both normal B cells and primary DLBCLs, prompting speculation that PTPROt might be a target of BCL6-mediated transcriptional repression. We next analyzed the PTPROt promoter region in silico and identified 3 candidate BCL6 binding sites. Using chromatin immunoprecipitation assays in BCR-type DLBCL cell lines, we demonstrated that BCL6 binding sites in the PTPROt promoter were occupied in vivo by the transcription factor. To assess the role of BCL6 in regulating PTPROt transcription, we cotransfected a luciferase reporter vector driven by the PTPROt promoter with constructs encoding either wild-type BCL6 or one of two inactive BCL6 mutants. Wild-type BCL6 suppressed PTPROt-driven luciferase activity in a dose-dependent manner whereas neither BCL6 mutant altered luciferase levels. Consistent with these observations, individual or combined mutations of the BCL6 binding sites in the PTPROt promoter reduced or abolished the response to BCL6. Next, we asked whether PTPROt was a physiologic target of BCL6 in normal B cells. Forced overexpression of BCL6 in normal naïve B cells was associated with a marked reduction in PTPROt transcript abundance. Since PTPROt functions as a SYK phosphatase, we then assessed the consequences of BCL6 depletion on SYK Y352 phosphorylation and BCR signaling in BCR-type DLBCL cell lines. BCL6 siRNA increased PTPROt transcript abundance 10-fold whereas other components of the BCR signaling pathway were unchanged. In cells transduced with BCL6 siRNA, tonic and anti-Ig associated phosphorylation of SYK Y352 and BLNK Y84 were significantly lower than in parental or mock-transduced cells. Taken together, these studies indicate that BCL6 represses the expression of the SYK phosphatase, PTPROt, and augments SYK-dependent tonic BCR signaling. Since BCL6 and SYK are both promising rational therapeutic targets in many DLBCLs, combined inhibition of these functionally related pathways warrants further study.

Blood, Nov 16, 2004

The Bcl-6 transcriptional repressor is required for the establishment of germinal centers during ... more The Bcl-6 transcriptional repressor is required for the establishment of germinal centers during B-cell maturation. Regulatory elements of the BCL6 gene are frequently mutated in diffuse large B-cell lymphomas (DLBCL), leading to inappropriately timed expression of Bcl-6. However, it is unknown whether Bcl-6 is involved in lymphomagenesis. Within Bcl-6, the N-terminal BTB domain is involved in mediating transcriptional repression. We investigated the mechanism of action of the Bcl-6 BTB domain using X-ray crystallography and functional assays. We found that the SMRT, N-CoR and BCoR corepressors bind directly to Bcl-6 through a 16 residue “BBD motif”, which fits into a highly specific “lateral groove” surface feature of the Bcl-6 BTB domain. To determine the contribution of BTB lateral groove recruitment of corepressors to transcriptional and biological functions of Bcl-6, we engineered small Bcl-6 BTB peptide inhibitors (BPI). BPI penetrate cells and localize to the nuclei where they specifically bound to Bcl-6 and blocked its interaction and co-localization with co-repressors. This resulted in dose-dependent blockade of repression by Bcl-6 in reporter assays and reactivation of endogenous Bcl-6 target genes in Bcl-6 expressing B-cells measured by real-time PCR. Loss of repression was caused by disruption of the endogenous Bcl-6 transcriptional repression complex. We found that BPI specifically excluded corepressors from the promoters of endogenous Bcl-6 target genes, resulting in a switch from repressed to activated histone code settings with consequent gene reactivation. From the biological standpoint, BPI injection into mice reproduced the B-cell phenotype of Bcl-6 null animals, as germinal center formation in response to T-cell dependent antigens was abrogated. To address the question of whether Bcl-6 is required to maintain the malignant phenotype of DLBCL, a panel of Bcl-6 positive and negative DLBCL cells were exposed to BPI. BPI induced growth suppression, cell cycle arrest and apoptosis in Bcl-6 positive but not negative DLBCL cells. Expression array analysis showed immediate upregulation of checkpoint genes involved in proliferation and apoptosis, allowing us to identify novel direct Bcl-6 target genes. In contrast, BPI did not upregulate differentiation related genes and did not induce differentiation in lymphoma cells, indicating that the Bcl-6 BTB domain lateral groove controls proliferation and survival pathways but not differentiation. Finally, BPI completely suppressed growth of Bcl-6 positive human DLBCL xenotransplants without any toxicity to other organs. In summary, specific disruption of the Bcl-6 BTB domain repression mechanism allowed us to reveal the contribution of specific corepressor recruitment to the activities of Bcl-6 in B-cells and to demonstrate that Bcl-6 is in fact an oncogene, required to maintain the malignant phenotype of Bcl-6 positive DLBCL. Our pre-clinical studies indicate that Bcl-6 is a bona fide therapeutic target and that BPI is a specific, potent and non-toxic anti-lymphoma therapeutic agent.

Life Sciences, 2021

The challenge in classical Hodgkin Lymphoma (cHL) management is the 30-40% of refractory/relapsed... more The challenge in classical Hodgkin Lymphoma (cHL) management is the 30-40% of refractory/relapsed cases.

HemaSphere, 2019

ying)) database to obtain data. We included cases diagnosed with lymphoma between 2006 and 2015 a... more ying)) database to obtain data. We included cases diagnosed with lymphoma between 2006 and 2015 at site related to orbital cavity C69.0-C69.9. Results: A total of 2340 cases were identified with a median age of 65.2. Females and patients of white race represented the majority of the analyzed cohort (n = 1249, 53.4%; n = 1846, 78.9%, respectively). Age-adjusted Incidence rate was 3 per 10,000. 5-years observed survival was 82 % (95% CI: 79.8%-84%) while the 5-years relative survival was 94.3 % (95% CI: 91.3%-96.2%). The most common pathology of studied cases was extranodal marginal zone lymphoma of mucosal-associated lymphoid tissue (MALT) (n = 1310, 56%) [Table 1]. Median survival was not reached at the study cutoff (Figure 1).

International Journal of Oncology, 2001

LP07 is a new cell line derived from P07 lung tumor, spontaneously arisen in a BALB/c mouse. LP07... more LP07 is a new cell line derived from P07 lung tumor, spontaneously arisen in a BALB/c mouse. LP07 is composed of heterogeneous epithelioid polyhedric cells that proliferate at a slow rate, have low plating efficiency and are unable to grow in soft agar. Only some LP07 cells expressed cytokeratins while most of them were positive for vimentin. Ultrastructure studies showed that LP07 cells established rudimentary intercellular unions, formed glandular-like conducts and presented conspicuous secretory granules, suggesting an epithelial-glandular origin, with neuroendocrine components. Upon injection LP07 cells formed poorly differentiated non-invasive adenocarcinomas, and tumor bearing mice developed leukocytosis, hypercalcemia and cachexia. This tumor cell line constitutes a useful tool to study lung tumor biology and paraneoplastic syndromes.

Blood, 2009

362 To identify genes requiered for the proliferation and survival of Diffuse Large B Cell Lympho... more 362 To identify genes requiered for the proliferation and survival of Diffuse Large B Cell Lymphomas (DLBCL) we conducted an “Achilles Heel” RNA interference screen in cell lines model of ABC (Activated B cell-like) and GCB (Germinal Center B-cell like) DLBCL subtypes. One of the most toxic small hairpins RNAs (shRNAs) in this screen targeted Oct2, encoding a POU domain transcriptional activator. Unlike Oct1, which is constitutively expressed in many cell types, Oct2 is primarily lymphoid restricted. It was identified by virtue of its ability to bind the highly conserved DNA octamer motif (ATGCAAAT) within immunoglobulin (Ig) genes promoters. The B cell specific co-activator OCA-B interacts with the POU domain of the octamer binding proteins enhancing their transactivation potential. Although Oct2 and OCA-B are not essential for Ig transcription, they are required for germinal center (GC) B cell differentiation. To understand the massive apoptotic cell death of DLBCL cells following...

Cancers, 2020

The Epstein–Barr virus (EBV), Kaposi sarcoma herpesvirus (KSHV) and human T-lymphotropic virus (H... more The Epstein–Barr virus (EBV), Kaposi sarcoma herpesvirus (KSHV) and human T-lymphotropic virus (HTLV-1) are lymphomagenic viruses with region-specific induced morbidity. The RIAL-CYTED aims to increase the knowledge of lymphoma in Latin America (LA), and, as such, we systematically analyzed the literature to better understand our risk for virus-induced lymphoma. We observed that high endemicity regions for certain lymphomas, e.g., Mexico and Peru, have a high incidence of EBV-positive lymphomas of T/NK cell origin. Peru also carries the highest frequency of EBV-positive classical Hodgkin lymphoma (HL) and EBV-positive diffuse large B cell lymphoma, not otherwise specified (NOS), than any other LA country. Adult T cell lymphoma is endemic to the North of Brazil and Chile. While only few cases of KSHV-positive lymphomas were found, in spite of the close correlation of Kaposi sarcoma and the prevalence of pathogenic types of KSHV. Both EBV-associated HL and Burkitt lymphoma mainly affe...

Cancer Research, 2015

Epidemiological studies have suggested a close link between obesity and breast cancer. There is a... more Epidemiological studies have suggested a close link between obesity and breast cancer. There is an immediate need to investigate the potential pathways linking obesity and breast cancer to have an early diagnosis in patients and optimize the chance of cure. FGF21 is a regulator of local and systemic metabolic homeostasis and its expression is induced in response to diverse physiological or pathological stressors. High serum levels of FGF21 were found in obese individuals, subjects with metabolic syndrome, type 2 diabetes mellitus and coronary heart disease. Up to date, the clinical implication of FGF21 in cancer was not elucidated. Our aim was to study the role of serum FGF21 as a diagnostic biomarker of breast cancer. The serum levels of FGF21 in 45 breast cancer women patients (median age 59, range 32-88 years) and 51 age-matched healthy controls were evaluated using a quantitative ELISA test (R&D Systems, Inc.). Patients’ samples [Stage (S) SI: 17; SII: 17; SIII: 6; ND: 5] were o...

Cancer Research, 2010

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC To identify genes r... more Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC To identify genes required for the proliferation and survival of Diffuse Large B Cell Lymphomas (DLBCL) we conducted an “Achilles Heel” RNA interference screen in cell lines model of ABC (Activated B Cell-like) and GCB (Germinal Center B-cell like) DLBCL subtypes. One of the most toxic shRNAs in this screen targeted Oct2, encoding a POU domain transcriptional activator, primarily lymphoid restricted. It was identified due its ability to bind the DNA octamer motif (ATGCAAAT) within immunoglobulin (Ig) genes promoters. The B cell specific coactivator OCA-B interacts with Oct-2 enhancing its transactivation potential. Although Oct2 and OCA-B are dipensable for Ig transcription, they are essential for germinal center (GC) development. To understand the apoptotic cell death of DLBCL cells following shRNA Oct2 induction we investigated the genetic pathways controlled by Oct2. We profiled gene expression changes in DLBCL cells upon Oct2 knocked down and merged this data with genome-wide assessment of Oct2 and OCA-B binding sites, coupling chromatin immunoprecipitation (ChiP) with high-throughput sequencing (ChIPSeq). More than 60% of the Oct2 target genes also showed OCA-B biding. The Oct2/OCA-B overlapping set of targets was enriched for genes selectively expressed in GC B cells. We found that Oct2/OCA-B lie upstream many transcription factors known to play critical roles in GC development such as BCL6, MTA3, PU.1, IRF8 and SpiB. Oct2 regulates these genes in DLBCL cells and in centroblasts. Among Oct-2 downstream effectors, BCL6 could rescue DLBCL cells from the Oct2 shRNA lethal effect. The Oct2/OCA-B binding of BCL6 promoter was confirmed by single locus ChIP in primary GC B-cells and derived lymphomas. Gel shifts experiments showed that Oct2 binds a non canonical octamer site at the BCL6 transcription start site. Our findings uncovered a novel aspect of the Oct2 biology believed to regulate the activity of octamer containing promoters. Interestingly, Oct2 controls the expression of GC specific genes that do not harbor a canonical octamer motif. By array CGH, amplification of Oct2 and OCA-B was found in less than 1% of non Hodgkin lymphoma (NHL) patient samples. Nonetheless, lymphoma cells become dependent on the Oct2 controlled network to survive, being an example of non oncogene addiction. This turns Oct2 into an attractive therapeutic target for NHL treatment. Oct2 and OCA-B lie upstream of BCL6, widely considered a master regulator of the GC response, suggesting that Oct2 directed therapy should kill the same DLBCLs as BCL6 Peptide Inhibitor. Furthermore, all GC and Post-GC B cells tested require Oct2 for survival, indicating that Oct2 directed therapy might have a broder activity spectrum than the BCL6 directed therapy. The Oct2/OCA-B binding interface would be amenable to attack with potential manageable toxicity since this interaction is exclusively required in GC B cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3898.

Cancer Research, 2020

Classic Hodgkin Lymphoma (cHL) is a germinal center derived lymphoma with 9,000 new cases diagnos... more Classic Hodgkin Lymphoma (cHL) is a germinal center derived lymphoma with 9,000 new cases diagnosed each year in the US. Despite 70-80% early stage cHL patients respond to current systemic treatment, 10% of patients are refractory to first-line therapy and 30-40% relapse. Refractory and relapsed disease is currently the challenge when treating cHL patients. There are no biomarkers suitable to identify patients that would be refractory or that could relapse at the primary diagnosis. Furthermore, there is no specific therapy rather than rescue chemotherapy schemes, which fails in 50% of the cases and associates with high risk severe toxicity. This highlights the need to deeper understand the cHL molecular biology and the screening for prognosis biomarkers and therapeutic innovative approaches. We have previously reported that the alternative NFkB pathway, mediated by Rel-B and NIK (NFkB Inducing Kinase), plays an important role in cHL survival. Its constitutive activation sustains hig...

Blood, 2005

The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas... more The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas and is required for establishment of germinal centers by normal B-cells. However, the mechanisms by which BCL6 licenses germinal center formation and lymphomagenesis are unknown. To characterize this mechanism we identified BCL6 target genes by expression arrays and high throughput chromatin immunoprecipitations. Remarkably, a number of these target genes were critical mediators of DNA damage sensing checkpoints including ATR and p53. Therefore, we hypothesized that BCL6 could attenuate DNA damage sensing by silencing these genes, which is likely a critical attribute for survival and proliferation of germinal center B-cells cells undergoing somatic hypermutation (SHM) and class switch recombination (CSR). Accordingly, we found that expression of BCL6 in normal diploid fibroblasts could block cellular sensing of DNA damage as demonstrated by loss of histone 2AX (H2AX) phosphorylation and...

Blood, 2005

The BCL6 (B-Cell-Lymphoma-6) oncoprotein is required for formation of germinal centers by normal ... more The BCL6 (B-Cell-Lymphoma-6) oncoprotein is required for formation of germinal centers by normal B-cells and is frequently constitutively activated in B-cell lymphomas. BCL6 is a transcriptional repressor of the BTB/POZ - zinc finger family of proteins. Transcriptional repressors are generally believed to function by binding to specific DNA sequences in target promoters and then recruiting a cohort of corepressor proteins that physically alter the chromatin structure of the locus leading to silencing. BCL6 can recruit several different corepressors. Accordingly, N-CoR, SMRT and BCoR bind to the BTB domain of BCL6, the NuRD complex binds to the second repression domain and ETO binds to the C-terminal zinc finger domain. These proteins recruit additional corepressors such as HDACS, thus forming multi-protein complexes. BCL6 is involved in both licensing germinal center B-cells for survival as well as blocking differentiation to memory or plasma cells. However, we found that blockade o...

Blood, 2019

Classic Hodgkin Lymphoma (cHL) is a germinal center derived lymphoma with 8,500-9,000 new cases/y... more Classic Hodgkin Lymphoma (cHL) is a germinal center derived lymphoma with 8,500-9,000 new cases/year diagnosed in the US. Despite 90% stage I cHL patients can respond to current systemic therapy, this drops to 60%, when diagnosed in advanced stages. Furthermore, 20-30% of diagnosed patients, would be refractory or would relapse and have a poor prognosis. Refractory and relapsed disease (RRD) is currently the challenge when treating cHL patients. There is no specific therapy to offer rather than rescue chemotherapy schemes, which fails in 50% of the cases and associates with high risk severe toxicity. This highlights the need to deeper understand the cHL molecular biology, the screening for molecular markers suitable to identify the risk of refractory and relapse disease and specific therapeutic directed-targets. We have previously reported that the alternative NFkB pathway, mediated by Rel-B and NIK (NFkB Inducing Kinase), plays an important role in cHL survival. Its constitutive ac...

Blood, 2018

Despite 90% stage I Hodgkin Lymphoma (HL) patients can respond to current systemic therapy, this ... more Despite 90% stage I Hodgkin Lymphoma (HL) patients can respond to current systemic therapy, this drops to 60%, when diagnosed in advanced stages. Nevertheless and independently of the lymphoma stage, the real challenge when treating these patients, is the refractory and relapsed disease. There is no molecular biomarker to identify patients that would be non-responsive to conventional treatment or that would relapse. Furthermore, rescue chemotherapy schemes for refractory and relapsed patients, associate with acute and late toxicity high risk. This highlights the need to deeper understand the HL molecular biology and the screening for predictive biomarkers as well as potential therapeutic directed-targets. We have previously reported that HL relies on the alternative NFkB pathway, mediated by RelB and NIK, to survive. Depletion of either RelB or NIK by shRNAs or pharmacological NIK inhibitors induce HL cell death. ChIP-Seq analysis uncovered RelB target genes showing RelB bound to BC...

Blood, 2011

428 NFkB is a pleitropic transcription factor (TF) known to play major roles in diverse cell proc... more 428 NFkB is a pleitropic transcription factor (TF) known to play major roles in diverse cell processes such as proliferation, apoptosis and inflammation. The REL or NFkB family is comprised of hetero or homo dimeric combinations of five members: RELA (p65), RELB, c-REL, NFKB1 (p50) and NFkB2 (p52). Despite considerable evidence supporting the role of the REL members in the immune system and lymphomagenesis, it has never been accurately established whether specific NFkB dimers control a particular set of target genes that account for the biological functions known to be mediated by these TFs. Furthermore, it is not clear whether NFkB controls different targets in distinct subsets of germinal center (GC) B cell derived lymphomas. We performed loss of function experiments delivering specific shRNAs to knock down each REL member in lymphoma cell lines modeling sequential stages of the GC B cell maturation: Mantle Cell Lymphoma (MCL), Diffuse Large B Cell Lymphoma [DLBCL including ABC-Li...

Blood, 2006

The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas... more The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas and is required for establishment of germinal centers (GCs) by normal B-cells. However, the mechanisms by which BCL6 licenses GC formation and lymphomagenesis remain unknown. To characterize this mechanism we identified BCL6 target genes through several methods. Most notable among these was ATR, a master DNA damage response mediator. We showed that primary BCL6-expressing GC centroblasts purified from human tonsils do not express ATR, do not activate downstream targets of ATR (Chk1) and exhibit an overall attenuated DNA damage checkpoint response (as shown in COMET assays, H2AX phosphorylation assays, and other methods). ATR expression, the activation of ChK1, and the DNA damage phenotype were fully rescued by BCL6 loss of function (induced by shRNA or by a specific BCL6 inhibitor that we designed). BCL6 expressing DLBCL cell lines exhibited the same BCL6-dependent DNA damage response p...

Journal of Cancer Research and Clinical Oncology, 2019

Purpose-Despite considerable evidence that supports the NF-kB role in the immune system and lymph... more Purpose-Despite considerable evidence that supports the NF-kB role in the immune system and lymphomagenesis, it is unclear whether specific NF-kB dimers control a particular set of genes that account for their biological functions. Our previous work showed that Hodgkin Lymphoma (HL) is unique, among germinal center (GC)-derived lymphomas, with respect to its dependency on Rel-B to survive. In contrast, diffuse large B-Cell lymphoma (DLBCL) including both Activated B-Cell-Like and Germinal Center B-Cell-Like, requires cREL and Rel-A to survive and it is not affected by Rel-B depletion. These findings highlighted the activity of specific NF-kB subunits in different GC-derived lymphomas. Methods-Sequenced chromatin immunoprecipitated DNA fragments (ChIP-Seq) analysis revealed an extensive NF-kB DNA-binding network in DLBCL and HL. The ChIP-Seq data was merged with microarray analysis following the Rel-A, Rel-B or cRel knockdown to determine effectively regulated genes. Results-Downstream target analysis showed enrichment for cell cycle control, among other signatures. Rel-B and cRel controlled different genes within the same signature in HL and DLBCL, respectively. BCL2 was exclusively controlled by Rel-B in HL. Both mRNA and protein

Clinical Research (Excluding Clinical Trials), 2019

This study discusses sociocultural changes in society in relation to parks by examining changes i... more This study discusses sociocultural changes in society in relation to parks by examining changes in government policies, the construction of port cities and parks, and characteristics of parks in Hokkaido before opening Otaru park and Temiya park located in Otaru city as a case study. Based on changes in society, historic document records were used to public and government perception toward opening and renovating Otaru Park and Temiya Park. Thus, the historical meanings of the parks could be established in terms of space and time, based on four parameters, namely, regional administration / residents' works, features required for park functioning, geographical characteristics of the parks, and the feature of the city when the parks were built. Further examination is required to improve parameters and data collection method for understanding the historical meanings of the parks from sociocultural perspective more deeply.

Blood, Nov 16, 2006

The BCL6 transcriptional repressor is the most commonly involved oncogene in diffuse large B-cell... more The BCL6 transcriptional repressor is the most commonly involved oncogene in diffuse large B-cell lymphomas (DLBCL). Constitutive expression of BCL6 has been proposed to mediate lymphomagenesis through several mechanisms, including evasion of cell death, proliferation and differentiation blockade. We show here that BCL6 mediates these effects through distinct mechanisms. First, we show that blocking the association of the SMRT corepressor with BCL6 using our specific peptide inhibitor (BPI) abrogates only the survival effects of BCL6 but has no effect on differentiation. Accordingly, BPI upregulates survival genes such as ATR and p53, but not genes associated with differentiation such as Blimp1, XBP and Syndecan. In contrast, BCL6 shRNA upregulates both survival and differentiation genes and induces both cell death and differentiation. We and others have shown that BCL6 can also directly bind to the MTA3 corepressor, which is implicated in differentiation of Burkitt lymphoma cells. We found that BCL6 and MTA3 are co-expressed in DLBCL cells and primary human centroblasts (the precursor cell for most DLBCLs). The endogenous BCL6 and MTA3 proteins interacted in DLBCLs cells in co-immunoprecipitation experiments. In contrast to SMRT blockade with BPI, siRNA depletion of MTA3 induced expression of the Blimp1, XBP and Syndecan but not p53 and ATR. MTA3 depletion induced plasmacytic differentiation within 72 hours as shown in functional assays and by surface markers. We performed ChIP on chip using custom arrays densely tiling with oligonucleotides covering the entire genomic loci of 20 BCL6 target genes. Interestingly, BCL6 formed different types of repression complexes at differentiation genes (Complex with MTA3/NuRD) vs. survival genes (complex with SMRT and N-CoR). BCL6-mediated repression of genes involved in survival and differentiation thus depend on distinct biochemical mechanisms. The relevance of these findings for human disease was underscored by the fact that we found a statistically significant positive correlation between MTA3 and BCL6 gene expression in a database of 176 human DLBCLs…

Cancer Research, Apr 15, 2006

Proc Amer Assoc Cancer Res, Volume 47, 2006 5651 The BCL6 (B-Cell-Lymphoma-6) transcriptional rep... more Proc Amer Assoc Cancer Res, Volume 47, 2006 5651 The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas and is required for establishment of germinal centers by normal B-cells. However, the mechanisms by which BCL6 licenses germinal center formation and lymphomagenesis are unknown. To characterize this mechanism we identified BCL6 target genes by expression arrays and high throughput chromatin immunoprecipitations. Remarkably, a number of these target genes were critical mediators of DNA damage sensing checkpoints including ATR and p53. Therefore, we hypothesized that BCL6 could attenuate DNA damage sensing by silencing these genes, which is likely a critical attribute for survival and proliferation of germinal center B-cells undergoing somatic hypermutation (SHM) and class switch recombination (CSR). Accordingly, we found that expression of BCL6 in normal diploid fibroblasts could block cellular sensing of DNA damage as demonstrated by loss of histone 2AX (H2AX) phosphorylation and delayed repair of double strand breaks, shown by COMET assays. Repression of ATR (but not p53 or other targets) was required for this phenotype. This is a physiological effect since the same result was observed when BCL6 was expressed in purified primary human tonsilar naive B-cells. Reciprocally, shRNA knockdown of BCL6 in B-cell lymphomas rescued repression of ATR, enhanced H2AX phosphorylation and accelerated repair of double strand breaks, independent of the status of p53. shRNA knockdown of BCL6 caused a marked increase of apoptosis in lymphoma cells in response to DNA damage, due to restored DNA damage checkpoint functions. Importantly, BCL6 knockdown had an identical effect on ATR levels, H2AX phosphorylation, DNA damage, and survival in purified primary human germinal center centroblasts. These results suggest that a major role of BCL6 in germinal center formation is to attenuate cellular response to DNA damage occurring as a byproduct of CSR and SHM. The same mechanism also seems to be required for lymphomagenesis, since we observed that sustained BCL6 expression in human primary mature B-cells leads to aberrant survival properties and genomic instability. Moreover, BCL6 blockade using a specific inhibitor molecule designed by our lab induces apoptosis in lymphoma cells and synergizes with DNA damaging agents. Therefore, we have identified a critical mechanism of action of the BCL6 oncoprotein in normal and pathogenic states and show that specific targeting of BCL6 could synergize with chemotherapy drugs for the therapy of B-cell lymphomas.

Blood, Nov 16, 2008

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease in which subsets of tumors likel... more Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease in which subsets of tumors likely rely upon different survival pathways. We previously identified a subset of primary DLBCLs with increased abundance of multiple components of the BCR signaling cascade including the spleen tyrosine kinase, SYK (“BCR-type” tumors). In related studies, we found that BCR-type DLBCL cell lines and primary tumors exhibited tonic B-cell receptor signaling. Since BCR-associated SYK activation initiates downstream events and amplifies the original BCR signal, we previously evaluated the efficacy of targeted SYK inhibition. Inhibition of SYK-dependent tonic BCR signaling induced apoptosis in the majority of “BCR-type” cell lines and primary DLBCLs. Furthermore, an oral SYK inhibitor (FosD) had promising activity in a subset of relapsed/refractory DLBCLs in a recently completed phase I/II clinical trial. “BCR-type” tumors also have more abundant expression of the transcriptional repressor, BCL6, and more frequent BCL6 translocations, greater repression of BCL6 target genes and increased sensitivity to BCL6 inhibitors. Since the same subset of primary DLBCLs relies upon SYK-dependent BCR signaling and BCL6-mediated transcriptional repression, we evaluated potential connections between BCL6 and SYK. SYK is a major substrate of the tissue-specific and developmentally regulated protein tyrosine phosphatase, PTPROt. For this reason, we first assessed the relative expression of BCL6 and PTPROt in two large independent series of transcriptionally profiled primary DLBCLs and two independent groups of highly purified normal B-cell subpopulations (naïve, germinal center, memory). There was a highly significant reciprocal pattern of expression of PTPROt and BCL6 in both normal B cells and primary DLBCLs, prompting speculation that PTPROt might be a target of BCL6-mediated transcriptional repression. We next analyzed the PTPROt promoter region in silico and identified 3 candidate BCL6 binding sites. Using chromatin immunoprecipitation assays in BCR-type DLBCL cell lines, we demonstrated that BCL6 binding sites in the PTPROt promoter were occupied in vivo by the transcription factor. To assess the role of BCL6 in regulating PTPROt transcription, we cotransfected a luciferase reporter vector driven by the PTPROt promoter with constructs encoding either wild-type BCL6 or one of two inactive BCL6 mutants. Wild-type BCL6 suppressed PTPROt-driven luciferase activity in a dose-dependent manner whereas neither BCL6 mutant altered luciferase levels. Consistent with these observations, individual or combined mutations of the BCL6 binding sites in the PTPROt promoter reduced or abolished the response to BCL6. Next, we asked whether PTPROt was a physiologic target of BCL6 in normal B cells. Forced overexpression of BCL6 in normal naïve B cells was associated with a marked reduction in PTPROt transcript abundance. Since PTPROt functions as a SYK phosphatase, we then assessed the consequences of BCL6 depletion on SYK Y352 phosphorylation and BCR signaling in BCR-type DLBCL cell lines. BCL6 siRNA increased PTPROt transcript abundance 10-fold whereas other components of the BCR signaling pathway were unchanged. In cells transduced with BCL6 siRNA, tonic and anti-Ig associated phosphorylation of SYK Y352 and BLNK Y84 were significantly lower than in parental or mock-transduced cells. Taken together, these studies indicate that BCL6 represses the expression of the SYK phosphatase, PTPROt, and augments SYK-dependent tonic BCR signaling. Since BCL6 and SYK are both promising rational therapeutic targets in many DLBCLs, combined inhibition of these functionally related pathways warrants further study.

Blood, Nov 16, 2004

The Bcl-6 transcriptional repressor is required for the establishment of germinal centers during ... more The Bcl-6 transcriptional repressor is required for the establishment of germinal centers during B-cell maturation. Regulatory elements of the BCL6 gene are frequently mutated in diffuse large B-cell lymphomas (DLBCL), leading to inappropriately timed expression of Bcl-6. However, it is unknown whether Bcl-6 is involved in lymphomagenesis. Within Bcl-6, the N-terminal BTB domain is involved in mediating transcriptional repression. We investigated the mechanism of action of the Bcl-6 BTB domain using X-ray crystallography and functional assays. We found that the SMRT, N-CoR and BCoR corepressors bind directly to Bcl-6 through a 16 residue “BBD motif”, which fits into a highly specific “lateral groove” surface feature of the Bcl-6 BTB domain. To determine the contribution of BTB lateral groove recruitment of corepressors to transcriptional and biological functions of Bcl-6, we engineered small Bcl-6 BTB peptide inhibitors (BPI). BPI penetrate cells and localize to the nuclei where they specifically bound to Bcl-6 and blocked its interaction and co-localization with co-repressors. This resulted in dose-dependent blockade of repression by Bcl-6 in reporter assays and reactivation of endogenous Bcl-6 target genes in Bcl-6 expressing B-cells measured by real-time PCR. Loss of repression was caused by disruption of the endogenous Bcl-6 transcriptional repression complex. We found that BPI specifically excluded corepressors from the promoters of endogenous Bcl-6 target genes, resulting in a switch from repressed to activated histone code settings with consequent gene reactivation. From the biological standpoint, BPI injection into mice reproduced the B-cell phenotype of Bcl-6 null animals, as germinal center formation in response to T-cell dependent antigens was abrogated. To address the question of whether Bcl-6 is required to maintain the malignant phenotype of DLBCL, a panel of Bcl-6 positive and negative DLBCL cells were exposed to BPI. BPI induced growth suppression, cell cycle arrest and apoptosis in Bcl-6 positive but not negative DLBCL cells. Expression array analysis showed immediate upregulation of checkpoint genes involved in proliferation and apoptosis, allowing us to identify novel direct Bcl-6 target genes. In contrast, BPI did not upregulate differentiation related genes and did not induce differentiation in lymphoma cells, indicating that the Bcl-6 BTB domain lateral groove controls proliferation and survival pathways but not differentiation. Finally, BPI completely suppressed growth of Bcl-6 positive human DLBCL xenotransplants without any toxicity to other organs. In summary, specific disruption of the Bcl-6 BTB domain repression mechanism allowed us to reveal the contribution of specific corepressor recruitment to the activities of Bcl-6 in B-cells and to demonstrate that Bcl-6 is in fact an oncogene, required to maintain the malignant phenotype of Bcl-6 positive DLBCL. Our pre-clinical studies indicate that Bcl-6 is a bona fide therapeutic target and that BPI is a specific, potent and non-toxic anti-lymphoma therapeutic agent.

Life Sciences, 2021

The challenge in classical Hodgkin Lymphoma (cHL) management is the 30-40% of refractory/relapsed... more The challenge in classical Hodgkin Lymphoma (cHL) management is the 30-40% of refractory/relapsed cases.

HemaSphere, 2019

ying)) database to obtain data. We included cases diagnosed with lymphoma between 2006 and 2015 a... more ying)) database to obtain data. We included cases diagnosed with lymphoma between 2006 and 2015 at site related to orbital cavity C69.0-C69.9. Results: A total of 2340 cases were identified with a median age of 65.2. Females and patients of white race represented the majority of the analyzed cohort (n = 1249, 53.4%; n = 1846, 78.9%, respectively). Age-adjusted Incidence rate was 3 per 10,000. 5-years observed survival was 82 % (95% CI: 79.8%-84%) while the 5-years relative survival was 94.3 % (95% CI: 91.3%-96.2%). The most common pathology of studied cases was extranodal marginal zone lymphoma of mucosal-associated lymphoid tissue (MALT) (n = 1310, 56%) [Table 1]. Median survival was not reached at the study cutoff (Figure 1).

International Journal of Oncology, 2001

LP07 is a new cell line derived from P07 lung tumor, spontaneously arisen in a BALB/c mouse. LP07... more LP07 is a new cell line derived from P07 lung tumor, spontaneously arisen in a BALB/c mouse. LP07 is composed of heterogeneous epithelioid polyhedric cells that proliferate at a slow rate, have low plating efficiency and are unable to grow in soft agar. Only some LP07 cells expressed cytokeratins while most of them were positive for vimentin. Ultrastructure studies showed that LP07 cells established rudimentary intercellular unions, formed glandular-like conducts and presented conspicuous secretory granules, suggesting an epithelial-glandular origin, with neuroendocrine components. Upon injection LP07 cells formed poorly differentiated non-invasive adenocarcinomas, and tumor bearing mice developed leukocytosis, hypercalcemia and cachexia. This tumor cell line constitutes a useful tool to study lung tumor biology and paraneoplastic syndromes.

Blood, 2009

362 To identify genes requiered for the proliferation and survival of Diffuse Large B Cell Lympho... more 362 To identify genes requiered for the proliferation and survival of Diffuse Large B Cell Lymphomas (DLBCL) we conducted an “Achilles Heel” RNA interference screen in cell lines model of ABC (Activated B cell-like) and GCB (Germinal Center B-cell like) DLBCL subtypes. One of the most toxic small hairpins RNAs (shRNAs) in this screen targeted Oct2, encoding a POU domain transcriptional activator. Unlike Oct1, which is constitutively expressed in many cell types, Oct2 is primarily lymphoid restricted. It was identified by virtue of its ability to bind the highly conserved DNA octamer motif (ATGCAAAT) within immunoglobulin (Ig) genes promoters. The B cell specific co-activator OCA-B interacts with the POU domain of the octamer binding proteins enhancing their transactivation potential. Although Oct2 and OCA-B are not essential for Ig transcription, they are required for germinal center (GC) B cell differentiation. To understand the massive apoptotic cell death of DLBCL cells following...

Cancers, 2020

The Epstein–Barr virus (EBV), Kaposi sarcoma herpesvirus (KSHV) and human T-lymphotropic virus (H... more The Epstein–Barr virus (EBV), Kaposi sarcoma herpesvirus (KSHV) and human T-lymphotropic virus (HTLV-1) are lymphomagenic viruses with region-specific induced morbidity. The RIAL-CYTED aims to increase the knowledge of lymphoma in Latin America (LA), and, as such, we systematically analyzed the literature to better understand our risk for virus-induced lymphoma. We observed that high endemicity regions for certain lymphomas, e.g., Mexico and Peru, have a high incidence of EBV-positive lymphomas of T/NK cell origin. Peru also carries the highest frequency of EBV-positive classical Hodgkin lymphoma (HL) and EBV-positive diffuse large B cell lymphoma, not otherwise specified (NOS), than any other LA country. Adult T cell lymphoma is endemic to the North of Brazil and Chile. While only few cases of KSHV-positive lymphomas were found, in spite of the close correlation of Kaposi sarcoma and the prevalence of pathogenic types of KSHV. Both EBV-associated HL and Burkitt lymphoma mainly affe...

Cancer Research, 2015

Epidemiological studies have suggested a close link between obesity and breast cancer. There is a... more Epidemiological studies have suggested a close link between obesity and breast cancer. There is an immediate need to investigate the potential pathways linking obesity and breast cancer to have an early diagnosis in patients and optimize the chance of cure. FGF21 is a regulator of local and systemic metabolic homeostasis and its expression is induced in response to diverse physiological or pathological stressors. High serum levels of FGF21 were found in obese individuals, subjects with metabolic syndrome, type 2 diabetes mellitus and coronary heart disease. Up to date, the clinical implication of FGF21 in cancer was not elucidated. Our aim was to study the role of serum FGF21 as a diagnostic biomarker of breast cancer. The serum levels of FGF21 in 45 breast cancer women patients (median age 59, range 32-88 years) and 51 age-matched healthy controls were evaluated using a quantitative ELISA test (R&D Systems, Inc.). Patients’ samples [Stage (S) SI: 17; SII: 17; SIII: 6; ND: 5] were o...

Cancer Research, 2010

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC To identify genes r... more Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC To identify genes required for the proliferation and survival of Diffuse Large B Cell Lymphomas (DLBCL) we conducted an “Achilles Heel” RNA interference screen in cell lines model of ABC (Activated B Cell-like) and GCB (Germinal Center B-cell like) DLBCL subtypes. One of the most toxic shRNAs in this screen targeted Oct2, encoding a POU domain transcriptional activator, primarily lymphoid restricted. It was identified due its ability to bind the DNA octamer motif (ATGCAAAT) within immunoglobulin (Ig) genes promoters. The B cell specific coactivator OCA-B interacts with Oct-2 enhancing its transactivation potential. Although Oct2 and OCA-B are dipensable for Ig transcription, they are essential for germinal center (GC) development. To understand the apoptotic cell death of DLBCL cells following shRNA Oct2 induction we investigated the genetic pathways controlled by Oct2. We profiled gene expression changes in DLBCL cells upon Oct2 knocked down and merged this data with genome-wide assessment of Oct2 and OCA-B binding sites, coupling chromatin immunoprecipitation (ChiP) with high-throughput sequencing (ChIPSeq). More than 60% of the Oct2 target genes also showed OCA-B biding. The Oct2/OCA-B overlapping set of targets was enriched for genes selectively expressed in GC B cells. We found that Oct2/OCA-B lie upstream many transcription factors known to play critical roles in GC development such as BCL6, MTA3, PU.1, IRF8 and SpiB. Oct2 regulates these genes in DLBCL cells and in centroblasts. Among Oct-2 downstream effectors, BCL6 could rescue DLBCL cells from the Oct2 shRNA lethal effect. The Oct2/OCA-B binding of BCL6 promoter was confirmed by single locus ChIP in primary GC B-cells and derived lymphomas. Gel shifts experiments showed that Oct2 binds a non canonical octamer site at the BCL6 transcription start site. Our findings uncovered a novel aspect of the Oct2 biology believed to regulate the activity of octamer containing promoters. Interestingly, Oct2 controls the expression of GC specific genes that do not harbor a canonical octamer motif. By array CGH, amplification of Oct2 and OCA-B was found in less than 1% of non Hodgkin lymphoma (NHL) patient samples. Nonetheless, lymphoma cells become dependent on the Oct2 controlled network to survive, being an example of non oncogene addiction. This turns Oct2 into an attractive therapeutic target for NHL treatment. Oct2 and OCA-B lie upstream of BCL6, widely considered a master regulator of the GC response, suggesting that Oct2 directed therapy should kill the same DLBCLs as BCL6 Peptide Inhibitor. Furthermore, all GC and Post-GC B cells tested require Oct2 for survival, indicating that Oct2 directed therapy might have a broder activity spectrum than the BCL6 directed therapy. The Oct2/OCA-B binding interface would be amenable to attack with potential manageable toxicity since this interaction is exclusively required in GC B cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3898.

Cancer Research, 2020

Classic Hodgkin Lymphoma (cHL) is a germinal center derived lymphoma with 9,000 new cases diagnos... more Classic Hodgkin Lymphoma (cHL) is a germinal center derived lymphoma with 9,000 new cases diagnosed each year in the US. Despite 70-80% early stage cHL patients respond to current systemic treatment, 10% of patients are refractory to first-line therapy and 30-40% relapse. Refractory and relapsed disease is currently the challenge when treating cHL patients. There are no biomarkers suitable to identify patients that would be refractory or that could relapse at the primary diagnosis. Furthermore, there is no specific therapy rather than rescue chemotherapy schemes, which fails in 50% of the cases and associates with high risk severe toxicity. This highlights the need to deeper understand the cHL molecular biology and the screening for prognosis biomarkers and therapeutic innovative approaches. We have previously reported that the alternative NFkB pathway, mediated by Rel-B and NIK (NFkB Inducing Kinase), plays an important role in cHL survival. Its constitutive activation sustains hig...

Blood, 2005

The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas... more The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas and is required for establishment of germinal centers by normal B-cells. However, the mechanisms by which BCL6 licenses germinal center formation and lymphomagenesis are unknown. To characterize this mechanism we identified BCL6 target genes by expression arrays and high throughput chromatin immunoprecipitations. Remarkably, a number of these target genes were critical mediators of DNA damage sensing checkpoints including ATR and p53. Therefore, we hypothesized that BCL6 could attenuate DNA damage sensing by silencing these genes, which is likely a critical attribute for survival and proliferation of germinal center B-cells cells undergoing somatic hypermutation (SHM) and class switch recombination (CSR). Accordingly, we found that expression of BCL6 in normal diploid fibroblasts could block cellular sensing of DNA damage as demonstrated by loss of histone 2AX (H2AX) phosphorylation and...

Blood, 2005

The BCL6 (B-Cell-Lymphoma-6) oncoprotein is required for formation of germinal centers by normal ... more The BCL6 (B-Cell-Lymphoma-6) oncoprotein is required for formation of germinal centers by normal B-cells and is frequently constitutively activated in B-cell lymphomas. BCL6 is a transcriptional repressor of the BTB/POZ - zinc finger family of proteins. Transcriptional repressors are generally believed to function by binding to specific DNA sequences in target promoters and then recruiting a cohort of corepressor proteins that physically alter the chromatin structure of the locus leading to silencing. BCL6 can recruit several different corepressors. Accordingly, N-CoR, SMRT and BCoR bind to the BTB domain of BCL6, the NuRD complex binds to the second repression domain and ETO binds to the C-terminal zinc finger domain. These proteins recruit additional corepressors such as HDACS, thus forming multi-protein complexes. BCL6 is involved in both licensing germinal center B-cells for survival as well as blocking differentiation to memory or plasma cells. However, we found that blockade o...

Blood, 2019

Classic Hodgkin Lymphoma (cHL) is a germinal center derived lymphoma with 8,500-9,000 new cases/y... more Classic Hodgkin Lymphoma (cHL) is a germinal center derived lymphoma with 8,500-9,000 new cases/year diagnosed in the US. Despite 90% stage I cHL patients can respond to current systemic therapy, this drops to 60%, when diagnosed in advanced stages. Furthermore, 20-30% of diagnosed patients, would be refractory or would relapse and have a poor prognosis. Refractory and relapsed disease (RRD) is currently the challenge when treating cHL patients. There is no specific therapy to offer rather than rescue chemotherapy schemes, which fails in 50% of the cases and associates with high risk severe toxicity. This highlights the need to deeper understand the cHL molecular biology, the screening for molecular markers suitable to identify the risk of refractory and relapse disease and specific therapeutic directed-targets. We have previously reported that the alternative NFkB pathway, mediated by Rel-B and NIK (NFkB Inducing Kinase), plays an important role in cHL survival. Its constitutive ac...

Blood, 2018

Despite 90% stage I Hodgkin Lymphoma (HL) patients can respond to current systemic therapy, this ... more Despite 90% stage I Hodgkin Lymphoma (HL) patients can respond to current systemic therapy, this drops to 60%, when diagnosed in advanced stages. Nevertheless and independently of the lymphoma stage, the real challenge when treating these patients, is the refractory and relapsed disease. There is no molecular biomarker to identify patients that would be non-responsive to conventional treatment or that would relapse. Furthermore, rescue chemotherapy schemes for refractory and relapsed patients, associate with acute and late toxicity high risk. This highlights the need to deeper understand the HL molecular biology and the screening for predictive biomarkers as well as potential therapeutic directed-targets. We have previously reported that HL relies on the alternative NFkB pathway, mediated by RelB and NIK, to survive. Depletion of either RelB or NIK by shRNAs or pharmacological NIK inhibitors induce HL cell death. ChIP-Seq analysis uncovered RelB target genes showing RelB bound to BC...

Blood, 2011

428 NFkB is a pleitropic transcription factor (TF) known to play major roles in diverse cell proc... more 428 NFkB is a pleitropic transcription factor (TF) known to play major roles in diverse cell processes such as proliferation, apoptosis and inflammation. The REL or NFkB family is comprised of hetero or homo dimeric combinations of five members: RELA (p65), RELB, c-REL, NFKB1 (p50) and NFkB2 (p52). Despite considerable evidence supporting the role of the REL members in the immune system and lymphomagenesis, it has never been accurately established whether specific NFkB dimers control a particular set of target genes that account for the biological functions known to be mediated by these TFs. Furthermore, it is not clear whether NFkB controls different targets in distinct subsets of germinal center (GC) B cell derived lymphomas. We performed loss of function experiments delivering specific shRNAs to knock down each REL member in lymphoma cell lines modeling sequential stages of the GC B cell maturation: Mantle Cell Lymphoma (MCL), Diffuse Large B Cell Lymphoma [DLBCL including ABC-Li...

Blood, 2006

The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas... more The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas and is required for establishment of germinal centers (GCs) by normal B-cells. However, the mechanisms by which BCL6 licenses GC formation and lymphomagenesis remain unknown. To characterize this mechanism we identified BCL6 target genes through several methods. Most notable among these was ATR, a master DNA damage response mediator. We showed that primary BCL6-expressing GC centroblasts purified from human tonsils do not express ATR, do not activate downstream targets of ATR (Chk1) and exhibit an overall attenuated DNA damage checkpoint response (as shown in COMET assays, H2AX phosphorylation assays, and other methods). ATR expression, the activation of ChK1, and the DNA damage phenotype were fully rescued by BCL6 loss of function (induced by shRNA or by a specific BCL6 inhibitor that we designed). BCL6 expressing DLBCL cell lines exhibited the same BCL6-dependent DNA damage response p...

Journal of Cancer Research and Clinical Oncology, 2019

Purpose-Despite considerable evidence that supports the NF-kB role in the immune system and lymph... more Purpose-Despite considerable evidence that supports the NF-kB role in the immune system and lymphomagenesis, it is unclear whether specific NF-kB dimers control a particular set of genes that account for their biological functions. Our previous work showed that Hodgkin Lymphoma (HL) is unique, among germinal center (GC)-derived lymphomas, with respect to its dependency on Rel-B to survive. In contrast, diffuse large B-Cell lymphoma (DLBCL) including both Activated B-Cell-Like and Germinal Center B-Cell-Like, requires cREL and Rel-A to survive and it is not affected by Rel-B depletion. These findings highlighted the activity of specific NF-kB subunits in different GC-derived lymphomas. Methods-Sequenced chromatin immunoprecipitated DNA fragments (ChIP-Seq) analysis revealed an extensive NF-kB DNA-binding network in DLBCL and HL. The ChIP-Seq data was merged with microarray analysis following the Rel-A, Rel-B or cRel knockdown to determine effectively regulated genes. Results-Downstream target analysis showed enrichment for cell cycle control, among other signatures. Rel-B and cRel controlled different genes within the same signature in HL and DLBCL, respectively. BCL2 was exclusively controlled by Rel-B in HL. Both mRNA and protein

Clinical Research (Excluding Clinical Trials), 2019

This study discusses sociocultural changes in society in relation to parks by examining changes i... more This study discusses sociocultural changes in society in relation to parks by examining changes in government policies, the construction of port cities and parks, and characteristics of parks in Hokkaido before opening Otaru park and Temiya park located in Otaru city as a case study. Based on changes in society, historic document records were used to public and government perception toward opening and renovating Otaru Park and Temiya Park. Thus, the historical meanings of the parks could be established in terms of space and time, based on four parameters, namely, regional administration / residents' works, features required for park functioning, geographical characteristics of the parks, and the feature of the city when the parks were built. Further examination is required to improve parameters and data collection method for understanding the historical meanings of the parks from sociocultural perspective more deeply.