Immunoglobulin Heavy Chain Enhancer Research Articles

Are genomic translocations predictable? (Retrospective on DOI 10.1002/bies.201100122).

It is sometimes useful for biologists to be reminded that the rules governing the physical universe apply to biological systems. Thermodynamic principles stipulate that rates for chemical reactions are proportional to the concentration(s) of reactants, a basic rule that applies equally to inorganic chemical reactions as well as to complex genomic rearrangements such as translocation and recombination. Jerome Dejardin recently predicted that genomic translocation and recombination in pathologic circumstances are facilitated by alterations in chromatin that permit aberrant transcription factor binding [1]. Transcription factor binding is posited to result in co-localization of non-adjacent loci in three dimensional space within the nucleus. This transcription-factor driven colocalization increases local concentration of the reacting species for translocation or recombination, thus accelerating genomic rearrangement. This prediction, while simple in concept, is powerful in terms of predicting sites of aberrant genomic transactions: they are predicted to be concentrated in the vicinity of binding sites of transcription factors found in the cell type(s) of interest and to occur at loci with locally accessible chromatin features. Issues relevant to Dejardin’s predictions have recently been addressed experimentally by a pair of elegant studies. Roukos et al. [2] examined translocation in an engineered system utilizing ultrahigh-throughput imaging that permits real-time monitoring of DNA ends (the reactants) within nuclei. Double-strand breaks were induced at two distinct (and uniquely marked) genetic loci by a restriction enzyme. The resulting DNA ends sampled nuclear space, occasionally finding and pairing with a translocation partner. Importantly, the distance between the two induced double strand breaks prior to end joining was critical for the frequency of translocation: ends closer to each other prior to pairing were more likely to translocate. This study suggests that an important component of the prediction by Dejardin is likely to be true, i.e. increasing the proximity of DNA double-strand breaks to each other (increasing the local concentration of reactants) leads to a higher likelihood of translocation. While this study supports the ‘concentration’ component of Dejardin’s prediction, it does not speak to the nature of the chromatin surrounding such an event. Evidence speaking for the role of chromatin in the generation of translocations was recently published by Hakim and colleagues [3]. This study defined the nuclear interaction partners of enhancers of immunoglobulin heavy chain (the well characterized Igh enhancers Eμ and Eα) in activated B lymphocytes using chromosome conformation capture (4C) experiments. The interactome of these transcription factor binding sites was enriched in active chromatin marked by high levels of histone acetylation, RNA polymerase occupancy, and active transcription. Introduction of DSBs at Igh (using a restriction enzyme) resulted in productive translocation to regions with similar chromatin features. This study indicates that translocation events occur with higher frequency at regions that are in close proximity in three dimensional space and that bear local chromatin features consistent with increased accessibility – precisely as predicted. The predictions of Dejardin – that translocation and recombination may be driven by transcription factor binding, colocalization of non-adjacent loci, and alterations in chromatin features – appear to be in strong agreement with newly emerging experimental data. It remains to be determined whether all features of the prediction agree with experimental data. The field awaits the emergence of additional studies directed at understanding these genomic rearrangements that may shed new light on the ability of this model to predict their location.

The relevance of testing the efficacy of anti-angiogenesis treatments on cells derived from primary tumors: a new method for the personalized treatment of renal cell carcinoma.

Despite the numerous available drugs, the most appropriate treatments for patients affected by common or rare renal cell carcinomas (RCC), like those associated with the Xp11.2 translocation/transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3) gene fusion (TFE3 RCC), are not clearly defined. We aimed to make a parallel between the sensitivity to targeted therapies on living patients and on cells derived from the initial tumor. Three patients diagnosed with a metastatic RCC (one clear cell RCC [ccRCC], two TFE3 RCC) were treated with anti-angiogenesis drugs. The concentrations of the different drugs giving 50% inhibition of cell proliferation (IC50) were determined with the Thiazolyl Blue Tetrazolium Bromide (MTT) assay on cells from the primary tumors and a reference sensitive RCC cell line (786-O). We considered the cells to be sensitive if the IC50 was lower or equal to that in 786-O cells, and insensitive if the IC50 was higher to that in 786-O cells (IC 50 of 6±1 µM for sunitinib, 10±1 µM for everolimus and 6±1 µM for sorafenib). Based on this standard, the response in patients and in cells was equivalent. The efficacy of anti-angiogenesis therapies was also tested in cells obtained from five patients with non-metastatic ccRCC, and untreated as recommended by clinical practice in order to determine the best treatment in case of progression toward a metastatic grade. In vitro experiments may represent a method for evaluating the best first-line treatment for personalized management of ccRCC during the period following surgery.

Tumour-promoting role of EMT-inducing transcription factor ZEB1 in mantle cell lymphoma

Tumour-promoting role of EMT-inducing transcription factor ZEB1 in mantle cell lymphoma

Role of the Igh intronic enhancer Eμ in clonal selection at the pre-B to immature B cell transition.

We previously described a checkpoint for allelic exclusion that occurs at the pre-B cell to immature B cell transition and is dependent upon the IgH intronic enhancer, Eμ. We now provide evidence that the breach in allelic exclusion associated with Eμ deletion results from decreased Igμ levels that make it difficult for emerging BCRs to reach the signaling threshold required for positive selection into the immature B cell compartment. We show that this compartment is smaller in mice carrying an Eμ-deficient, but functional, IgH allele (VHΔ(a)). Pre-B cells in such mice produce ≈ 50% wild-type levels of Igμ (mRNA and protein), and this is associated with diminished signals, as measured by phosphorylation of pre-BCR/BCR downstream signaling proteins. Providing Eμ-deficient mice with a preassembled VL gene led not only to a larger immature B cell compartment but also to a decrease in "double-producers," suggesting that H chain/L chain combinations with superior signaling properties can overcome the signaling defect associated with low Igμ-chain and can eliminate the selective advantage of "double-producers" that achieve higher Igμ-chain levels through expression of a second IgH allele. Finally, we found that "double-producers" in Eμ-deficient mice include a subpopulation with autoreactive BCRs. We infer that BCRs with IgH chain from the Eμ-deficient allele are ignored during negative selection owing to their comparatively low density. In summary, these studies show that Eμ's effect on IgH levels at the pre-B cell to immature B cell transition strongly influences allelic exclusion, the breadth of the mature BCR repertoire, and the emergence of autoimmune B cells.

Mammalian target of rapamycin pathway activity in alveolar soft part sarcoma

Alveolar soft part sarcoma (ASPS) is a distinct type of soft tissue sarcoma holding a specific ASPL-TFE3 fusion transcript. Curative therapy is based on surgical removal, whereas lately, antiangiogenic targeted therapy regimens have proven effective. In ASPS, analysis of small series additionally display mTOR (mammalian target of rapamycin) pathway activity, thus making mTOR a possible additive target in ASPS, because it is in other tumor entities. Therefore, we systematically evaluated mTOR pathway activity in a large series of ASPS in comparison with soft tissue sarcomas of other differentiation (non-ASPS). Upstream and downstream factors of mTOR signaling and ancillary targets were analyzed in 103 cases (22 ASPS, 81 non-ASPS) by immunohistochemistry mostly using phospho-specific antibodies. TFE3 (transcription factor for immunoglobulin heavy-chain enhancer 3) translocation status was determined by FISH and RT-PCR. All ASPS were positive in TFE3 break-apart FISH and exhibited specific fusion products when RNA was available (type 1: 9x, type 2: 11x), whereas TFE3-immunoreactive non-ASPS did not. In ASPS, TFE3-, cMET-, pAKT T308- (all P < .0001), pp70S6K- (P = .002), and p4EBP1 (P = .087) expression levels were elevated, whereas pAKT S473 was decreased (P < .0001). In addition, ASPS exhibited higher TFE3-, cMET-, pAKT T308-, and pp70S6K- expression levels compared with TFE3-immunopositive non-ASPS sarcomas (all P < .001). We demonstrate elevated mTOR complex 1 (mTORC1) activity in ASPS independent of mTOR complex 2 (mTORC2) activation. mTORC1 activity seems to be related to the existence of ASPL-TFE3 fusion transcripts because TFE3-immunoreactive non-ASPS without ASPL-TFE3 fusion transcripts exhibit significantly lower mTORC1 activation status. Small molecule-based targeting of mTOR might therefore represent a potential mechanism in ASPS alone or in combination with contemporary upstream approaches.

MMSET is the key molecular target in t(4;14) myeloma

The t(4;14)(p16.3;q32.3) is found in 15% of presenting multiple myeloma (MM) cases and is associated with a significantly worse prognosis than other biological subgroups. As a consequence of the translocation, two genes are aberrantly expressed, the fibroblast growth factor receptor 3 (FGFR3) and a multiple myeloma SET domain containing protein, MMSET (WHSC1/NSD2), both of which have potential oncogenic activity.1 Importantly, FGFR3 shows only weak transforming activity and is eventually lost in 30% of patients,2 suggesting that it is not the main oncogenic factor. In contrast, MMSET gene overexpression is universal, and when it is knocked down experimentally, there is inhibition of proliferation, induction of apoptosis and alteration of cell adhesion,3, 4, 5 suggesting it is central to the pathogenesis of this subtype of MM. MMSET is known to have histone methyl transferase activity6 and is deregulated early on in the genesis of developing myeloma, and could therefore constitute a good therapeutic target. The MMSET locus in t(4;14) myeloma patients has a complicated genomic structure and after translocation events and RNA splicing, a number of different transcripts are generated (Figure 1). This genetic complexity of MMSET has been added to recently by the discovery of the H/ACA box RNA ACA11 (SCARNA22), that has been found within intron 20 of MMSET and is also overexpressed in the t(4;14) subgroup.7 This small RNA has been suggested to be key to the pathogenesis of t(4;14) MM, raising the question that it may constitute the main therapeutic target.

The CCND1 c.870G&gt;A polymorphism is a risk factor for t(11;14)(q13;q32) multiple myeloma

A number of specific chromosomal abnormalities define the subgroups of multiple myeloma. In a meta-analysis of two genomewide association studies of multiple myeloma totaling 1,661 patients we investigated risk for developing a specific tumor karyotype. The t(11;14) (q13;q32) translocation in which CCDN1 is placed under the control of the immunoglobin heavy chain enhancer was strongly associated with the CCDN1 870G>A polymorphism (P =7.96 x10-11). These results provide for a model in which a constitutional genetic factor is associated with risk of a specific chromosomal translocation.

Effect of the 3�Regulatory Region insulation on B cell development and DNA rearrangements at the Immunoglobulin heavy chain locus

Event Abstract Back to Event Effect of the 3’Regulatory Region insulation on B cell development and DNA rearrangements at the Immunoglobulin heavy chain locus Fatima-Zohra Braikia1, Mohamed Moutahir1, Caroline Conte1* and Ahmed A. Khamlichi1 1 University of Toulouse, CNRS UMR5089/University of Toulouse, France The Immunoglobulin heavy chain locus (IgH) undergoes a series of developmentally-regulated DNA rearrangements, named V(D)J recombination. Regulation of these events is effected by various cis-acting elements among which the Eµ enhancer plays a major role. Another important cis-element is the 3’ Regulatory Region, located downstream of the IgH locus, and which was suggested to interact with Eµ enhancer. CTCF-binding elements have been described at the IgH locus and are thought to mediate the formation of large loops that may be important for various processes linked to V(D)J recombination. In this study, we have adopted an insulation approach by inserting the well-characterized cHS4 insulator of the chicken beta-globin locus, upstream of the IgH 3’RR. We generated two mouse lines in which either the wild-type, CTCF-binding, cHS4 insulator was used or a mutated version of it devoid of CTCF-binding site. We will present our recent results on the long-range effect of 3’RR insulation on B cell development, V(D)J recombination, allelic exclusion and IgH expression. Acknowledgements This work was funded by INCa (PLBIO11-181), ANR (ANR-07-BLAN-0080-03), ARC (Grant SFI20101201441), LNCC-Comité de Haute-Garonne, Cancéropôle GSO, and the University of Toulouse Keywords: B cell development, germline transcription, IgH enhancer, VDJ, CSR Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune receptors and signaling Citation: Braikia F, Moutahir M, Conte C and Khamlichi AA (2013). Effect of the 3’Regulatory Region insulation on B cell development and DNA rearrangements at the Immunoglobulin heavy chain locus. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00251 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 14 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Caroline Conte, University of Toulouse, CNRS UMR5089/University of Toulouse, Toulouse, France, caroline.conte@ipbs.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Fatima-Zohra Braikia Mohamed Moutahir Caroline Conte Ahmed A Khamlichi Google Fatima-Zohra Braikia Mohamed Moutahir Caroline Conte Ahmed A Khamlichi Google Scholar Fatima-Zohra Braikia Mohamed Moutahir Caroline Conte Ahmed A Khamlichi PubMed Fatima-Zohra Braikia Mohamed Moutahir Caroline Conte Ahmed A Khamlichi Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Deregulated Expression of HDAC9 in B-Cells Leads to Lymphoproliferative Disorders As Well As Germinal Center and Post-Germinal Center Derived Lymphomas

AbstractAbstract 3505Histone Deacetylase 9 (HDAC9) is a class IIa enzyme that, within the hematopoietic system, is preferentially expressed in the B-cell lineage. Previously we have observed that, compared with normal cellular counterparts, expression of HDAC9 appears deregulated in B-lymphoid tumors (Petrie et al., JBC 278: 16059, 2003 and unpublished results). To examine the role of HDAC9 in B-cell transformation we generated a mouse model that constitutively expresses human HDAC9 from early stages of B-cell development under the control of the immunoglobulin heavy chain (IgH) enhancer (Eμ). We demonstrate that from six months of age (6–12 months), Eμ-HDAC9 transgenic mice displayed splenomegaly in a statistically significant fraction of cases (3/17, 18%), which was associated with histopathologic and immunophenotypic evidence of lymphoproliferative disease (LPD) (p<0.0001). Later in life (12 to 23 months of age), 24% (19/78) of Eμ-HDAC9 mice developed a spectrum of mature clonal B-cell lymphoproliferations including monoclonal B-cell lymphocytosis (MBL, 12/78, 15%), splenic marginal zone lymphoma (SMZL, 4/78, 5%) and diffuse large B-cell lymphoma (DLBCL, 3/78, 4%). In contrast, a low background incidence of these malignancies (2%) was observed in control mice (p = 0.0008). Importantly, the HDAC9 gene is located within chromosomal region 7p21, which is a common target of frequent genomic gains in B-cell non-Hodgkin lymphoma (B-NHL) cases and B-NHL cell lines. Consistently, copy number gains in the 7p21.1 chromosomal region were found in 25/79 (32%) primary DLBCL cases analyzed (including one B-NHL patient with a copy number gain only within the 5’ end of the HDAC9 gene). Mechanism underlying the role of HDAC9 in lymphomagenesis appears to involve deacetylation of both p53 (leading to deactivation of p53) and BCL6 (leading to activation of BCL6), consistent with a role of a general hypoacetylated state in B-NHL pathogenesis, which is underscored by recently identified inactivating mutations in a number of key histone acetyltransferases. In summary, aberrant expression of HDAC9 leads to the development of B-cell lymphoproliferations, including germinal center (GC) and post-GC lymphomas, showing similarities with those occurring in humans. These findings have relevant therapeutic implications in view of current attempts to use HDAC inhibitors as anti-lymphoma drugs. Disclosures:No relevant conflicts of interest to declare.

MiR-221 and MiR-222 Patterns Characterize Burkitt Lymphoma in Human and Mouse Model

AbstractAbstract 1304Recently, a class of noncoding RNAs called microRNA (miRNAs) has emerged as critical gene regulators in cell growth, differentiation, disease and development. MiRNAs are 18–24 nucleotide long noncoding RNAs, which regulate gene expression by pairing with 3′ untranslated region (UTR) of target mRNA and inhibiting protein translation and/or inducing mRNA degradation. Deregulated miRNA expression is reported in various human diseases including lymphomas, suggesting an important role in their pathogenesis.According to WHO classification, Burkitt lymphoma (BL) is a rare, highly aggressive NHL composed of monomorphic medium-sized B cells with multiple nucleoli and numerous mitotic figures and is more common in children than in adults. The molecular feature of BL is the translocation that places MYC under the control of immunoglobulin gene regulatory elements. High levels of c-MYC have been clearly shown to have a tumour-promoting effect. However, there is recent evidence that infrequent cases may lack an identifiable MYC translocation, the explanation for which is still uncertain, though suggesting the existence of pathogenetic mechanisms alternative to genetic alterations.Over the past years miRNA signatures have been described to characterize and classify different types of BL or to investigate the expression of miRNAs possibly regulated by c-Myc in BL cases positive or negative for Myc translocation. However, it remained unclear the functional role of differentially expressed miRNAs and no further studies have been conducted. We performed miRNA expression profile to gain further insights into the molecular pathology of BL. We conducted array analysis on a set of 5 sporadic BL patients, 3 endemic BL patients, 9 reactive tissues and 11 cases of mononucleosis. Our profile is the first one that shows the different expression between BL cases and normal B cells whereas recent miRNA profiles have been conducted in BL compared to other B-NHL (B-CLL, MCL & FL). A common trend of miRNAs altered expression was also observed by NanoString analysis in 10 BL cell lines compared to 5 normal CD-19+ B cells. Among several miRNAs previously described be deregulated in BL we identified a severe down-regulation of miR-221, miR-222 in all classes of comparisons we analyzed. The down-regulation of miR-221 and miR-222 associated to BL has been also confirmed by q-RT-PCR method in a different cohort of BL patients (20) compared to the healthy controls (6). We found that interesting considering the up-regulation of miR-221 and miR-222 previously confirmed in a lot of solid tumors by multiple studies. We are investigating a different role of the cluster miR-222 and miR-221 in lymphomas that have a different process in carcinogenesis than solid tumors.In vivo models to study the lymphomagenesis of BL have been created but until now no one studied the importance of the miRNAs in vivo. We analyzed the expression of miR-221 and miR-222 in a Myc transgenic mouse model. The transgene construct consists of the Myc oncogene (c-myc) in association with the Emu immunoglobulin heavy chain enhancer and Myc promoter. Expression of the mouse Myc transgene is restricted to the B cell lineage. Previously it has been shown an increase of pre-B cells in the bone marrow throughout life of hemizygotes and a transient increase in large pre-B cells in the blood at 3–4 weeks of age; moreover spontaneous pre-B and B cell lymphomas reach an incidence of 50% at 15–20 weeks in hemizygous progeny of a wildtype female mated with a hemizygous male. We observed the development of Burkitt lymphoma within 10 weeks of birth in 14 out of 25 Eu-Myc transgenic mice and a premature death in 5 out or 25 transgenic mice within 6–8 weeks of birth without showing any enlarged lymph nodes. Transgenic mice with masses showed the same phenotype characterized by enlarged spleen (3 fold), lymphosarcomas associated with BL and enlarged lymph nodes around the neck area. B-cells have been negatively selected from enlarged lymph nodes and enlarged spleen. A qRT-PCR has been conducted to evaluate the miR-221 and miR-222 expression. The miRNA levels showed a down-regulation in B cells collected from the masses when compared to normal B cells derived from the spleen of WT mice.In conclusion, our study reveals new insights into the functional significance in loss of miR-221 and miR-222 expression in BL pathogenesis. Disclosures:No relevant conflicts of interest to declare.

Non-IG Aberrations of FOXP1 in B-Cell Malignancies Result in an Aberrant Expression of N-Truncated FOXP1 Isoforms.

AbstractAbstract 2411Forkhead Box P1 (FOXP1) is a member of the FOX family of transcription factors which play critical roles in immune responses, organ development and tumorigenesis. Multiple lines of evidence suggest that FOXP1 functions as an oncogene in B-cell neoplasms, being overexpressed in a variety of lymphomas and targeted by rare but recurrent translocations in marginal zone lymphoma (MZL) and diffuse large B-cell lymphoma (DLBCL). The most common translocation is t(3;14)(p13;q32) which brings FOXP1 under a transcriptional control of the IGH enhancers. Several cases with non-IG rearrangements of FOXP1 have been reported but thus far, not studied in details. Of note, Brown et al. (2008) demonstrated that FOXP1+ ABC-DLBCLs lacking FOXP1 rearrangements selectively express smaller N-truncated FOXP1 isoforms and subsequently hypothesized that the oncogenic role of FOXP1 is only exhibited by smaller isoforms.To unravel molecular consequences of non-IG aberrations of FOXP1 in lymphoma, we performed extensive FISH and molecular investigations of five cases harboring the rearrangements (index cases), complemented by studies of t(3;14)+ lymphoma and FOXP1+ and FOXP1-DLBCL/MZL. The index cases were presented with MZL, transforming MZL, CLL in Richter transformation and gastric DLBCL. The identified non-IG aberrations included t(2;3)(q36;p13), t(3;10)(p13;q24), inv(3)(p13q11) and an unknown IGH/IGK/IGL-negative translocation. One case displayed a microdeletion of FOXP1 spanning the region between exon 3 and 7, as shown by FISH and aCGH. The aberrations constituted a part of complex karyotypes and were subclonal in 3 cases. All tumors displayed a nuclear expression of the FOXP1 protein. FISH showed that the 3p13 breakpoints of index cases clustered within the coding region of FOXP1, contrasting with the 5’ end breakpoints of a classical t(3;14)/IGH-FOXP1. Further FISH analysis of 3 available cases mapped the reciprocal breakpoints within AP1S3 (2q36) located in an opposite transcriptional orientation to FOXP1, in the 10q24 region harboring 3 genes (TMEM180->, C10orf95<-, ACTRIA<-), and in a gene poor region at 3q11, respectively. The applied 5’Race PCR additionally identified the PLEKHG1/FOXP1 fusion in one case of FOXP1+DLBCL. The fusion, however, occurred out of the reading frame of FOXP1 and was not functional. The pattern of FOXP1 expression was initially investigated using qRT-PCR. Both t(3;14)+cases, one FOXP1+DLBCL and non-malignant lymph nodes revealed an upregulation of all coding exons analyzed (6–18), while the index cases and the remaining FOXP1+DLBCLs displayed a significant upregulation of exons 7–18. Further RNA-sequencing of two index cases, 6 FOXP1+DLBCLs and 2 FOXP1-DLBCLs confirmed a general pattern of FOXP1 detected by qRT-PCR, and additionally identified several expressed FOXP1 isoforms. Their composition and a relative abundance differed in particular cases, but the most frequently expressed was the full length transcript and 3 N-truncated isoforms annotated by Ensembl as FOXP1–004, −203 and −009. The highest expression of the full length transcript was observed in cases with t(3;14) and FOXP1+GC-DLBCL; in the remaining cases a selective expression of N-truncated isoforms was noted. Results of transcriptomic studies were validated by Western analysis.Altogether, we showed that non-IG aberrations of FOXP1 are recurrent in B-cell lymphoma. These rearrangements are heterogeneous (reciprocal translocations, inversion, internal deletion) but constantly affect the coding part of the gene. They do not generate chimeric genes but result in an aberrant expression of N-truncated FOXP1 isoforms. Molecular mechanisms underlying these aberrations are largely unknown. Our findings suggest a dual role of FOXP1 in the pathogenesis of lymphoma: the full length FOXP1 upregulated by a primary IGH-mediated t(3;14) is a potent transforming factor implicated in development of lymphoma, while the N-truncated FOXP1 isoforms generated and activated by secondary non-IG aberrations, play a role in lymphoma progression. Of note, the latter variants are also overexpressed in ABC-DLBCL. Further studies of the wild type FOXP1 protein and N-truncated isoforms are required to demonstrate their oncogenic potential in vitro and in vivo. Disclosures:No relevant conflicts of interest to declare.

Transcriptome Changes Induced by Epstein-Barr Virus LMP1 and LMP2A in Transgenic Lymphocytes and Lymphoma

ABSTRACTLatent membrane protein 1 (LMP1) and LMP2A affect cell growth in both epithelial cells and lymphocytes. In this study, the effects on cellular gene expression were determined by microarray analysis of transgenic mice expressing LMP1, LMP2A, or both using the immunoglobulin heavy chain promoter and enhancer. Large differential changes were detected, indicating that LMP1 and LMP2A can both potently affect host gene transcription, inducing distinct transcriptional profiles. Seventy percent of the changes detected in LMP1/2A doubly transgenic lymphocytes were also modulated by LMP1 or LMP2A alone. These common and unique expression changes indicate that the combined effects of LMP1 and LMP2A may be additive, synergistic, or inhibitory. Using significant pathway analysis, the expression changes detected in LMP1, LMP2A, and LMP1/2A transgenic B lymphocytes were predicted to commonly target cancer and inflammatory pathways. Additionally, using the correlation coefficient to calculate the regulation of known c-Rel and Stat3 transcriptional targets, both were found to be enhanced in LMP1 lymphocytes and lymphomas, and a selection of Stat3 targets was further evaluated and confirmed using quantitative reverse transcription-PCR (RT-PCR). Analyses of the effects on cell growth and viability revealed that LMP2A transgenic lymphocytes had the greatest enhanced viability in vitro; however, doubly transgenic lymphocytes (LMP1/2A) did not have enhanced survival in culture and these mice were similar to negative littermates. These findings indicate that the combined expression of LMP1 and LMP2A has potentially different biological outcomes than when the two proteins are expressed individually.

Immunoglobulin heavy chain enhancer's (Eμ) role in cell selection at the pre-B to immature B cell transition (109.7)

Abstract We have recently described a second check-point for allelic exclusion that operates at the pre-B to immature B cell transition and is dependent upon the IgH intronic enhancer Eμ. We suggest that Eμ’s function at this transition is to ensure that IgH chains are made at levels sufficient to signal positive selection, regardless of light chain sequences and despite mono-allelic expression of the heavy chain. Evidence leading to this hypothesis includes our findings that B cells expressing an Eμ-deficient allele undergo heightened light-chain editing (suggesting that only some light-chain partners can circumvent the effects of lower BCR levels), or express a second IgH allele (“double-producing” cells to achieve higher BCR levels). We now show that B cells expressing an Eμ-deficient IgH allele, consistent with inefficient positive selection, remain longer at the pre-B cell stage, resulting in a significantly diminished immature B cell compartment. To determine whether escape from this developmental stall is dependent upon light chain, we limited the available light chain repertoire in these animals by pre-assembled VL insertion. The result was an effect on both B cell development and the emergence of double-producing cells. Together with our earlier findings, these new results suggest that IgH levels at the pre-B to immature B transition have an important impact on the breadth of the BCR repertoire as well as on the maintenance of allelic exclusion.

TFE3 Rearrangements in Adult Renal Cell Carcinoma

Renal cell carcinoma (RCC) with chromosomal rearrangement of transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3) at Xp11.2 is a distinct subtype that was initially described in children and has been reported to display an indolent course. Recent reports have identified RCC with TFE3 rearrangements in adults and have suggested a more aggressive course in this population. However, only a few studies have examined these tumors in a large series of consecutively treated adults. We screened 632 RCCs from patients consecutively treated by surgery at a single institution by fluorescence in situ hybridization to detect TFE3 rearrangements. We identified 6 RCCs with TFE3 rearrangement. Patient ages ranged from 25 to 78 years and included 4 women and 2 men. Tumors showed significant histologic variability. Comparison of the clinical and pathologic features between RCCs with TFE3 rearrangements and RCCs without TFE3 rearrangements showed no significant differences. Follow-up period for patients with TFE3-rearranged RCC ranged from 0.8 to 16.5 years, with 4 of 6 dying from the disease. Cancer-specific survival for patients with TFE3-rearranged RCC was significantly worse than for patients with TFE3-rearrangement-negative papillary-type RCC (P<0.001) but not different from that for TFE3-rearrangement-negative clear cell-type RCC. In conclusion, we present an assessment of TFE3 rearrangement status in a large series of adults consecutively treated by surgery for RCC. Our findings confirm that RCCs with TFE3 rearrangement account for only approximately 1% of adult RCCs. The results also suggest that adult RCC with TFE3 rearrangement may be a clinically aggressive tumor.

BH3-only proteins in apoptosis at a glance

Apoptosis is a genetically programmed process for the elimination of damaged or redundant cells by activation of caspases (aspartate-specific cysteine proteases). Caspases cleave vital proteins, leading the cell to fragment into vesicles that are rapidly engulfed by phagocytes (for reviews, see [

Polymorphisms of the IgH enhancer HS1.2 and risk of systemic lupus erythematosus

ObjectiveTo determine whether the allelic frequency variation of the HS1.2 enhancer of the immunoglobulin heavy chain (IgH) 3′ regulatory region (3′RR-1) locus represents a risk factor for systemic lupus erythematosus...

A p53 Defect Sensitizes Various Stages of B Cell Development to Lymphomagenesis in Mice Carrying an IgH 3′ Regulatory Region-Driven c-myc Transgene

Although c-myc is classically described as the driving oncogene in Burkitt's lymphoma (BL), deregulation and mutations of c-myc have been reported in multiple solid tumors and in other mature B cell malignancies such as mantle cell lymphoma (MCL), myeloma, and plasma cell lymphoma (PCL). After translocation into the IgH locus, c-myc is constitutively expressed under the control of active IgH enhancers. Those located in the IgH 3' regulatory region (3'RR) are master control elements of class switch recombination and of the transcriptional burst associated with plasma cell differentiation. c-myc-3'RR mice are prone to lymphomas with rather homogeneous, most often BL-like, phenotypes with incomplete penetrance (75% tumor incidence) and long latencies (10-12 mo). To reproduce c-myc-induced mature B cell lymphomagenesis in the context of an additional defect often observed in human lymphomas, we intercrossed c-myc-3'RR with p53(+/-) mice. Double transgenic c-myc-3'RR/p53(+/-) mice developed lymphoma with short latency (2-4 mo) and full penetrance (100% tumor incidence). The spectrum of B lymphomas occurring in c-myc-3'RR/p53(+/-) mice was widened, including nonactivated (CD43(-)) BL, activated (CD43(+)) BL, MCL-like lymphoma, and PCL, thus showing that 3'RR-mediated deregulation of c-myc can promote various types of B lymphoproliferation in cells that first acquired a p53 defect. c-myc/p53(+/-) mice closely reproduce many features of BL, MCL, and PCL and provide a novel and efficient model to dissect the molecular events leading to c-myc-induced lymphomagenesis and an important tool to test potential therapeutic agents on malignant B cells featuring various maturation stages.

Inhibition of c-Myc Expression and Function in Hematologic Malignancies

Abstract 1409MYC is among the most prolific oncogenes in cancer, yet pharmacologic strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have undertaken to target c-Myc transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative co-activator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain-containing proteins as transcriptional regulatory factors for c-Myc. BET inhibition with JQ1 rapidly downregulates c-Myc transcription, followed by depletion of chromatin-bound c-Myc and genome-wide downregulation of Myc-dependent target genes. In translational model systems of multiple myeloma and Burkitt's lymphoma, both canonical MYC -dependent hematologic malignancies, JQ1 treatment produces a potent antiproliferative effect associated with cell cycle arrest. In multiple myeloma, adhesion to bone marrow stroma is associated with upregulation of BRD4, a BET bromomdomain coactivator protein. Inhibition of Myc function with JQ1 leads to impaired adhesion to stroma and cellular senescence, a classical Myc-specific phenotype. Mechanistically, JQ1 treatment depletes BRD4 from IgH enhancers, leading to prompt and robust downregulation of MYC transcription. In vivo efficacy of JQ1 in two disseminated models of multiple myeloma and in a Burkitt's lymphoma human xenograft establishes the therapeutic rationale for BET bromodomain inhibition in these diseases. Together, these studies identify a mechanistic rationale for targeting c-Myc in human cancer, and potentially other undruggable oncogenes driven by immunoglobulin rearrangement.Note: G.C.I. and J.E.D. have made equal contributions to this research; C.S.M. and J.E.B. are jointly senior authors Disclosures:Richardson:Millennium: Advisory Board; Celgene: Advisory Board; Johnson & Johnson: Advisory Board; Novartis: Advisory Board; Bristol Myers Squibb: Advisory Board. Ghobrial:Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celgene:; Novartis:. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Amnis Therapeutics: Consultancy, Honoraria; PharmaMar:; OSI Pharmaceuticals: Research Funding; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding. Bradner:Acetylon Pharmaceuticals: Scientific Founder; SHAPE Pharmaceuticals: Scientific Founder; Tensha Therapeutics: Scientific Founder.

FoxO Feedback Control of Basal IRS-2 Expression in Pancreatic β-Cells Is Distinct From That in Hepatocytes

OBJECTIVEAppropriate regulation of insulin receptor substrate 2 (IRS-2) expression in pancreatic β-cells is essential to adequately compensate for insulin resistance. In liver, basal IRS-2 expression is controlled via a temporal negative feedback of sterol regulatory element–binding protein 1 (SREBP-1) to antagonize transcription factors forkhead box class O (FoxO)1/FoxO3a at an insulin response element (IRE) on the IRS-2 promoter. The purpose of the study was to examine if a similar mechanism controlled IRS-2 expression in β-cells.RESEARCH DESIGN AND METHODSIRS-2 mRNA and protein expression, as well as IRS-2 gene promoter activity, were examined in isolated rat islets. Specific transcription factor association with the IRE on the IRS-2 promoter was examined by chromatin immunoprecipitation (ChIP) assay, and their nuclear translocation was examined by immunofluorescence. A direct in vivo effect of insulin on control of IRS-2 expression in liver and pancreatic islets was also investigated.RESULTSIn IRS-2 promoter-reporter assays conducted in isolated islets, removal of the IRE decreased basal IRS-2 promoter activity in β-cells up to 80%. Activation of IRS signaling in isolated rat islets by insulin/IGF-I (used as an experimental in vitro tool) or downstream constitutive activation of protein kinase B (PKB) significantly decreased IRS-2 expression. In contrast, inhibition of phosphatidylinositol 3-kinase (PI3K) or PKB significantly increased IRS-2 levels in β-cells. ChIP assays indicated that transcription factors FoxO1 and FoxO3a associated with the IRE on the IRS-2 promoter in β-cells in a PI3K/PKB–dependent manner, whereas others, such as SREBP-1, the transcription factor binding to immunoglobulin heavy chain enhancer 3′, and the aryl hydrocarbon receptor nuclear translocator (ARNT), did not. However, only FoxO3a, not FoxO1, was capable of driving IRS-2 promoter activity via the IRE in β-cells. In vivo studies showed insulin was able to suppress IRS-2 expression via activation of SREBP-1 in the liver, but this mechanism was not apparent in pancreatic islets from the same animal.CONCLUSIONSThe molecular mechanism for feedback control of IRS signaling to decrease IRS-2 expression in liver and β-cells is quite distinct, with a predominant role played by FoxO3a in β-cells.

Abstract B16: Tumor suppressive functions by ATF2/7 transcription factors in a cMyc-driven B-cell lymphoma model

Abstract ATF2 and the highly homologous ATF7 are members of the AP-1 transcription factor family which regulate expression of genes involved in development and stress response. In cancer, ATF2 has been reported to function both as a tumor suppressor and tumor promoter. The aim of the project was to investigate the possible roles of ATF2/7 in cMyc-driven B-cell lymphoma development and to characterize their interaction with the cMyc oncogene. Previously, cMyc, which is frequently deregulated in Burkitt's lymphoma (Rabbitts et al, 1984), was shown to prolong the half-life and induce phosphorylation of ATF2 (Miethe et al, 2001). In addition, the MAP kinase JNK and its substrate ATF2 are frequently found upregulated in B-cell lymphomas (Kersten et al, 2004; Gururajan et al, 2005). We established a mouse B-cell lymphoma model by crossing B-cell specific knockouts of Atf2/7 (Atf2ΔBC, Atf7−/−) with Eμ-Myc transgenic animals. Eμ-Myc mice express the cMyc proto-oncogene driven by the immunoglobulin heavy chain enhancer (Eμ) and develop B-cell lymphomas with high frequency. We compared timing of tumor onset between Eμ-myc, Atf2ΔBC, Atf7−/− Eμ-myc, Atf2ΔBC/+, Atf7−/−, and Eμ-myc, Atf7−/−. In addition, we derived cell lines fom Eμ-Myc-driven lymphomas and characterized growth, apoptosis, and drug sensitivity in the presence or absence of functional Atf2/7. Atf2 and its phosphorylated (activated) form were found to be upregulated in cells obtained from tumor samples from Eμ-Myc mice in comparison to normal B lymphocytes. Analysis of tumor-free survival showed that Eμ-myc, AtfΔBC, Atf7−/− develop B-cell lymphomas at significantly faster rates than controls. In vitro deletion of Atf2 in cell lines derived from cMyc-driven lymphomas resulted in a marked reduction in the basal levels of apoptosis. Furthermore, Atf2/7-deficient Eμ-myc tumor cells were significantly more resistant to genotoxic stress (e.g., doxorubicin) induced apoptosis. Gene expression analysis identified proapoptotic members of the Bcl-2 family as possible transcriptional targets of Atf2. In this study, we show a novel tumor suppressive role of ATF2 in cMyc-induced lymphomagenesis and suggest the potential functions of ATF2 in regulating programmed cell death. The results also imply that the evaluation of levels of activated ATF2 in clinical samples of B-cell lymphoma may give an indication of their sensitivity to chemotherapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B16.