Combined copy number and mutation analysis identifies oncogenic pathways associated with transformation of follicular lymphoma.

Follicular lymphoma (FL) is typically an indolent disease, but 30-40% of FL cases transform into an aggressive lymphoma (tFL) with a poor prognosis. To identify the genetic changes that drive this transformation, we sequenced the exomes of 12 cases with paired FL and tFL biopsies and identified 45 recurrently mutated genes in the FL-tFL data set and 39 in the tFL cases. We selected 496 genes of potential importance in transformation and sequenced them in 23 additional tFL cases. Integration of the mutation data with copy-number abnormality (CNA) data provided complementary information. We found recurrent mutations of miR-142, which has not been previously been reported to be mutated in FL/tFL. The genes most frequently mutated in tFL included KMT2D (MLL2), CREBBP, EZH2, BCL2 and MEF2B. Many recurrently mutated genes are involved in epigenetic regulation, the Janus-activated kinase-signal transducer and activator of transcription (STAT) or the nuclear factor-κB pathways, immune surveillance and cell cycle regulation or are TFs involved in B-cell development. Of particular interest are mutations and CNAs affecting S1P-activated pathways through S1PR1 or S1PR2, which likely regulate lymphoma cell migration and survival outside of follicles. Our custom gene enrichment panel provides high depth of coverage for the study of clonal evolution or divergence.

The miRNA-17∼92 cluster mediates chemoresistance and enhances tumor growth in mantle cell lymphoma via PI3K/AKT pathway activation.

The median survival of patients with mantle cell lymphoma (MCL) ranges from 3 to 5 years with current chemotherapeutic regimens. A common secondary genomic alteration detected in MCL is chromosome 13q31-q32 gain/amplification, which targets a microRNA (miRNA) cluster, miR-17∼92. On the basis of gene expression profiling, we found that high level expression of C13orf25, the primary transcript from which these miRNAs are processed, was associated with poorer survival in patients with MCL (P=0.021). We demonstrated that the protein phosphatase PHLPP2, an important negative regulator of the PI3K/AKT pathway, was a direct target of miR-17∼92 miRNAs, in addition to PTEN and BIM. These proteins were down-modulated in MCL cells with overexpression of the miR-17∼92 cluster. Overexpression of miR-17∼92 activated the PI3K/AKT pathway and inhibited chemotherapy-induced apoptosis in MCL cell lines. Conversely, inhibition of miR-17∼92 expression suppressed the PI3K/AKT pathway and inhibited tumor growth in a xenograft MCL mouse model. Targeting the miR-17∼92 cluster may therefore provide a novel therapeutic approach for patients with MCL.

BCL3 is induced by IL-6 via Stat3 binding to intronic enhancer HS4 and represses its own transcription.

BCL3 is a proto-oncogene affected by chromosomal translocations in some patients with chronic lymphocytic leukemia. It is an IkappaB family protein that is involved in transcriptional regulation of a number of NF-kappaB target genes. In this study, interleukin (IL)-6-induced BCL3 expression and its effect on survival of multiple myeloma (MM) cells were examined. We demonstrate the upregulation of BCL3 by IL-6 in INA-6 and other MM cell lines. Sequence analysis of the BCL3 gene locus revealed four potential signal transducer and activator of transcription (Stat) binding sites within two conserved intronic enhancers regions: one located within enhancer HS3 and three within HS4. Chromatin immunoprecipitation experiments showed increased Stat3 binding to both enhancers upon IL-6 stimulation. Silencing Stat3 expression by small interfering RNA (siRNA) abrogated BCL3 expression by IL-6. Using reporter gene assays, we demonstrate that BCL3 transcription depends on HS4. Mutation of the Stat motifs within HS4 abolished IL-6-dependent BCL3 induction. Furthermore, BCL3 transcription was inhibited by its own gene product. This repressive feedback is mediated by NF-kappaB sites within the promoter and HS3. Finally, we show that overexpression of BCL3 increases apoptosis, whereas BCL3-specific siRNA does not affect the viability of INA-6 cells suggesting that BCL3 is not essential for the survival of these cells.

Minimal structural elements of an inhibitory anti-ATF1/CREB single-chain antibody fragment (scFv41.4).

Antibody variable domains represent potential structural models for the rational design of therapeutic molecules that bind cellular proteins with high affinity and specificity. The Activating Transcription Factor 1 (ATF1)/Cyclic AMP Response Element Binding Protein (CREB) family of transcription factors are particularly relevant targets due to their strong association with melanoma and clear cell sarcoma. Biochemical and structural investigations were performed to optimize a single-chain antibody fragment (scFv), scFv41.4, that disrupts the binding of ATF1/CREB to cyclic-AMP response elements (CRE) in vitro and inhibits transcriptional activation in cells. Molecular modeling and ligand docking simulations suggested that scFv41.4 could function as a disulfide-deficient single domain scFv. Functional studies verified that deletion of the light chain did not result in reduced inhibitory activity. The isolated heavy chain was predicted to assume a relaxed structural conformation that maintained a functional antigen binding pocket. The minimal structural elements necessary for intracellular function were further analyzed by selective deletion of CDR1 and CDR2. V(H)-CDR1 and V(H)-CDR3 were shown to play a key role in antigen binding activity, but V(H)-CDR2 was dispensable. Thus, scFv41.4 represents a unique molecule with potential for use in the design of peptidomimetic derivatives having therapeutic application to human cancer.

Lymphadenopathy, splenomegaly, and altered immunoglobulin production in BCL3 transgenic mice.

The candidate proto-oncogene BCL3 was isolated through its involvement in the t(14;19) found in chronic lymphocytic leukemia and other B-cell neoplasms. As a member of the I kappaB family, BCL3 plays a role in the immune response by interactions with the NF-kappaB family of transcription factors. In order to study the role of BCL3 overexpression in lymphoid malignancies, we generated five lines of E mu-BCL3 transgenic mice. Transgenic animals develop normally but show splenomegaly and an accumulation of mature B cells in lymph nodes, bone marrow and peritoneal cavity. A hyperresponsive immune system is suggested by the follicular hyperplasia and plasmacytosis in lymph nodes of unimmunized animals, increased incidence of antibodies to self-antigens, and a heightened response to cross-linking of surface IgM. Statistically significant decreases in serum IgM and IgG3, but an increase in IgG1 and IgA were also observed. No lymphoid neoplasms have been identified in transgenic animals. The expansion of B cells in vivo is consistent with the overexpression of BCL3 as being one step in the multi-step process of leukemogenesis. The phenotype also suggests that BCL3 plays a part in B cell proliferation and isotype switching.

Replication of a common fragile site, FRA3B, occurs late in S phase and is delayed further upon induction: implications for the mechanism of fragile site induction.

The FRA3B at 3p14.2 is the most highly expressed of the common fragile sites observed when DNA replication is perturbed by aphidicolin or folate stress. The molecular basis for chromosome fragility at FRA3B is unknown. In contrast to the rare fragile sites, including FRAXA, no repeat motifs, such as trinucleotide repeats, have been identified within FRA3B. Several lines of evidence suggest that fragile sites are regions of DNA whose replication is unusually sensitive to interference. We have used fluorescence in situ hybridization to determine the relative timing of replication of FRA3B sequences. Our studies revealed that FRA3B sequences are late replicating. Exposure to aphidicolin, an inhibitor of both DNA polymerase alpha and delta, results in a reproducible delay in the timing of replication, and some cells enter G2without having completed replication of FRA3B sequences. Our results support a model in which common fragile sites are sequences that initiate replication late in S phase or are slow to replicate, and the chromosomal breaks and gaps observed in metaphase cells are due to unreplicated DNA.

An FHIT tumor suppressor gene?

The FRA3B at 3p14.2 is the most common of the constitutive aphidicolin-inducible fragile sites. Using independent approaches, four groups of investigators have cloned and characterized this fragile site. The results of these studies have revealed that the FRA3B differs from other heretofore cloned rare fragile sites. First, instability as manifested by chromosome breakage occurs over a large region of DNA, encompassing at least 500 kb. Second, sequence analysis has not revealed trinucleotide repeat motifs, characteristic of the rare fragile sites. In addition to containing the FRA3B, band 3p14 is also likely to contain a tumor suppressor gene, as evidenced by the presence of deletions, rearrangements, and allele loss in a variety of human tumors, including lung, renal, nasopharyngeal, cervical, and breast carcinomas. The recently cloned FHIT gene in 3p14.2 is a promising candidate tumor suppressor gene, since aberrant FHIT transcripts have been found in a significant proportion of cancer-derived cell lines and primary tumors of the digestive and respiratory tracts. Nonetheless, several lines of evidence garnered over the past year have called into question the role of FHIT as a classical tumor suppressor gene, and raised the question of whether its apparent involvement simply reflects its location within an unstable region of the genome. In the following study, we have summarized the evidence in support of FHIT as a tumor suppressor gene as well as evidence against such a role, and the experimental evidence needed to demonstrate that FHIT functions as a tumor suppressor gene in the pathogenesis of human tumors. The paradigm of FHIT emphasizes that confirming the role of a candidate tumor suppressor gene may prove difficult, particularly for those genes that are located in genetically unstable regions.

Molecular analysis of the t(8;14)(q24;q11) chromosomal breakpoint junctions in the T-cell leukemia line MOLT-16.

The MOLT-16 cell line was established from the leukemic cells of a patient with T-cell acute lymphoblastic leukemia and contains a t(8;14)(q24;q11) resulting in juxtaposition of sequences downstream of the MYC gene on chromosome 8 and the J region of the T-cell receptor alpha chain gene (TCRA) on chromosome 14. The reciprocal translocation involved a complex rearrangement with two chromosome breakpoints within the TCRAJ region on chromosome 14, resulting in inversion of a 1.4 kb DNA fragment between the two breakpoints. The 5' border of the inversion joints with another segment of chromosome 14, whereas the 3' border joins with a region of chromosome 8 located at least 257 kb downstream of MYC. Extensive deletions have occurred on both chromosomes 8 and 14 in conjunction with the translocation. To investigate the possible involvement of the V(D)J recombinase in this translocation, we analyzed the nucleotide sequences surrounding the translocation breakpoints. The breakpoint on chromosome 14 occurs between a segment coding for a TCRAJ sequence and its hepatamer-nonamer signal. Heptamer-nonamer consensus sequences are also identified on chromosome 8 adjacent to the breakpoint. Inserted N and P nucleotides are observed at the breakpoint junctions.

Precise localization of the FHIT gene to the common fragile site at 3p14.2 (FRA3B) and characterization of homozygous deletions within FRA3B that affect FHIT transcription in tumor cell lines.

Chromosomal or allelic losses at 3p14 are common in a variety of human tumors, including those of the lung, breast, kidney, and head and neck. This suggests the existence of a tumor suppressor gene in this band. A promising candidate is the recently cloned FHIT gene, which spans the common fragile site, FRA3B, at 3p14.2. We previously identified a region of fragility at 3p14.2 (FRA3B) of > 85 kb by cloning DNA flanking pSV2neo integrations and constructed a partial genomic contig of the region. Using probes from the contig, we tested for deletions within this region in DNA from 105 human tumor cell lines, predominantly derived from lung cancers. We identified one gastric and four lung cancer cell lines with homozygous interstitial deletions involving the FRA3B region. The deletion in one lung cancer cell line lies entirely within our contig and is < 65 kb. We have identified, cloned, and sequenced this breakpoint junction. We have also shown that our probes lie within intron S of the FHIT gene and, furthermore, that exon 5 is located approximately 1 kb from one of our probes and, thus, lies within the region of fragility. Two lines with entirely intronic deletions yield FHIT transcripts of normal size. In one of these, this was the sole transcript identified. In the other line, an FHIT transcript completely normal in sequence was accompanied by two larger abnormal transcripts. These results leave open the possibility that some homozygous deletions within the FHIT gene are without phenotypic effect and result from genetic instability of this region. However, taken together, our results provide evidence that breakage and rearrangement within the FRA3B fragile site sequences result in alterations of FHIT and are likely to be involved in carcinogenesis.

BCL3 rearrangements and t(14;19) in chronic lymphocytic leukemia and other B-cell malignancies: a molecular and cytogenetic study.

The t(14;19)(q32.3;q13.1) is a recurring translocation found in the neoplastic cells of some patients with chronic lymphocytic leukemia (CLL) or other B-lymphocytic neoplasms. We previously cloned the translocation breakpoint junctions present in the leukemic cells from three such patients and identified a gene, BCL3, whose transcription is increased as a result of the translocation. In the present paper, we describe three additional patients with the t(14;19), one with lymphoma and two with CLL, and report the cloning and sequencing of the breakpoint junction in one of these patients as well as in a previously reported patient. We and others have found that the breakpoints on chromosome 14, with one exception, fall within the switch region upstream of the immunoglobulin heavy chain C alpha 1 or C alpha 2 sequences. Several of the breaks within chromosome 19 fall immediately upstream of the BCL3 gene, but several others are more than 16 kb 5' of the gene. Most patients with CLL and the t(14;19) also show trisomy 12.

BCL6 can repress transcription from the human immunodeficiency virus type I promoter/enhancer region.

The gene BCL6 encodes a zinc finger protein with similarities to transcription factors. We previously reported that a number of viral genomes, including human immunodeficiency virus type I (HIV-1), contain sequences which are similar to the BCL6 DNA-binding consensus in their promoter regions. Electrophoretic mobility shift assays showed that the full-length BCL6 protein extracted from transfected COS cells and a bacterially expressed truncated protein containing the BCL6 zinc fingers can bind specifically to DNA from the U3 promoter/enhancer region of HIV-1. Transient transfections were performed to analyze the effects of the BCL6 protein on luciferase expression driven by the HIV-1 long terminal repeat (LTR) sequences. Full-length BCL6 significantly repressed luciferase activity compared with multiple controls. We conclude that the BCL6 protein can bind to the HIV-1 promoter-enhancer region and contains a domain upstream of its zinc fingers that can repress transcription from the HIV-1 LTR.

CpG-rich sequences close to the site of duplication within the human IGH constant region.

Human immunoglobulin heavy chains are encoded by a highly complex locus, IGH, which encompasses many transcriptional units, including nine alternative constant region genes. Much of the constant region gene cluster was duplicated during hominoid evolution. In rodents, IGH is known to be regulated by multiple elements, including several enhancers 3' of the alpha chain-encoding A constant region gene, CA, the last transcribed region. Sequences downstream of the two human CA genes, possibly containing homologous enhancer elements, have not yet been reported. By long-range mapping of genomic DNA, a cluster of unmethylated rare restriction sites, representing a potential CpG island, was previously reported downstream of each CA gene, close to the 3' end of the duplicated region. Such potential CpG islands are candidates for additional IGH regulatory elements. We isolated bacteriophage clones containing these clusters of methylation-sensitive restriction sites, which lie within short CpG-rich stretches. Some of these sites showed tissue-specific methylation. 3' of the unmethylated CpG-rich sequences, clones derived from the 5' (telomeric) copy of the duplicated region, contained, in order, endogenous retroviral sequences, a processed ELK1 pseudogene, and the pseudogene IGHGP. Comparison with the presumed 3' (centromeric) copy of the duplicated region showed that similarity was lost exactly at the end of the retroviral long terminal repeat sequences. These results imply that an endogenous retroviral integration was present immediately 3' of IGH in the common hominoid ancestor and suggest models for the duplication of the region.

Diverse effects of BCL3 phosphorylation on its modulation of NF-kappaB p52 homodimer binding to DNA.

IkappaB proteins control the subcellular localization and DNA binding activity of NF-kappaB transcription factors. BCL3 is a nuclear IkappaB that can inhibit or enhance the binding of NF-kappaB p50 or p52 homodimers to consensus DNA-binding (kappaB) sequences or form a kappaB-binding complex with homodimers. To study BCL3 function, we have used gel shift analysis and tagged protein and tagged DNA coprecipitation analyses. Our results show that at intermediate ratios of BCL3 to p52 all observed phosphoforms of BCL3 are able to form a kappaB-binding complex with p52 homodimers. At low BCL3/p52 ratios, BCL3 increases the rate of p52 homodimer binding to kappaB sites in the presence of nonconsensus DNA and dissociates from the complex. At high BCL3/p52 ratios, BCL3 forms a higher order inhibitory complex with p52 homodimers. All of these effects depend on BCL3 phosphorylation and relative concentration. These results indicate that BCL3 phosphorylation may affect its regulation of NF-kappaB-dependent transcription in vivo.

Complete lymphoid chimerism and chronic graft-versus-host disease in an infant recipient of a hepatic allograft from an HLA-homozygous parental living donor.

Living-related donor liver transplantation (LDLT) is an accepted approach to pediatric liver transplantation. Parental donation imposes a significant risk of chimerism with graft-versus-host disease (GVHD) because donors homozygous at all HLA loci (1.6% of the population) present no mismatched HLA antigens to be recognized by their offspring's immune system. The case of a 9-month-old who underwent LDLT with her 23-year-old HLA-homozygous mother as a donor demonstrates the consequences of this occurrence. The patient developed GVHD with aplastic anemia; the patient's nucleated peripheral blood elements were shown to be entirely derived from the donor. Later, after some marrow recovery, the patient's circulating lymphocytes had a donor origin, while the marrow-derived neutrophils had a recipient origin. The patient suffers from chronic GVHD and debilitating skin disease several years posttransplant. Our current protocol calls for HLA typing to eliminate parents who are homozygous at all HLA loci as donors of hepatic allografts to their children.

Direct cloning of DNA sequences from the common fragile site region at chromosome band 3p14.2.

Despite several lines of evidence suggesting that common chromosomal fragile sites are biologically important as hot spots for recombination, their structure remains unknown. We showed previously that the plasmid pSV2neo preferentially integrates into bands containing fragile sites in cells transfected under conditions of fragile site induction. Here we report the isolation and characterization of the DNA sequences from two such independent integrations into 3p14.2, a common fragile site (FRA3B). These FRA3B region sequences were shown to lie within a 1330-kb YAC, 850A6, approximately 350 kb telomeric of the breakpoint of t(3;8), a constitutional rearrangement. The two integration sites are 10 kb apart, but each integration is associated with a deletion. We have constructed a partial genomic contig of the integration sites and deleted regions spanning approximately 85 kb. Analysis of the DNA sequences immediately surrounding the plasmid integrations revealed no known coding sequences or repeat structures resembling the (CGG)n motif characteristic of the rare fragile sites. In addition, by Southern blotting analysis, none of the phage clones isolated from the FRA3B region were found to contain CGG repeats. Fluorescence in situ hybridization analysis of genomic clones from this contig to metaphase cells induced to express breaks demonstrated hybridization adjoining the chromosome breaks, and occasionally the hybridization signal spanned the break. The results imply that breakage occurs at variable positions within a large region (at least on the order of 85 kb). Together, these data suggest that the structure of FRA3B differs from that of rare fragile sites.

Resolution of Helicobacter pylori-associated gastric lymphoproliferative disease in a child.

A possible causative association between Helicobacter pylori infection and gastric lymphoproliferative disorders has recently been recognized. The case of a 14-year-old girl who was diagnosed with H. pylori gastritis and associated gastric lymphoproliferative disease of the low-grade mucosa-associated lymphoid tissue type is reported. The patient was treated only for the H. pylori infection (amoxicillin, bismuth, and metronidazole) without any adjuvant chemotherapy or surgery for her lymphoproliferative disorder. This treatment not only resulted in the eradication of the microorganism but also complete resolution of her lymphoproliferative disease. The patient was subsequently followed up for a period of 7 years. There has been no histological recurrence of H. pylori gastritis or gastric lymphoproliferative disease. It is believed that this is the first report to describe a long-term follow-up of an H. pylori-associated gastric lymphoproliferative disorder in a pediatric patient who was exclusively treated for H. pylori infection. The observations in this report suggest that H. pylori-associated low-grade gastric lymphoproliferative disease can be completely cured by eradicating the organism. Therefore, this therapeutic approach, combined with close follow-up, should be the treatment of choice in children with this associated condition before attempting more aggressive treatments, thus potentially avoiding chemotherapy and/or (partial) gastrectomy.

Kinetic proofreading in T-cell receptor signal transduction.

Like other cell-surface receptors with intrinsic or associated protein-tyrosine kinase activity, the T-cell receptor complex undergoes a number of modifications, including tyrosine phosphorylation steps, after ligand binding but before transmitting a signal. The requirement for these modifications introduces a temporal lag between ligand binding and receptor signaling. A model for the T-cell receptor is proposed in which this feature greatly enhances the receptor's ability to discriminate between a foreign antigen and self-antigens with only moderately lower affinity. The proposed scheme is a form of kinetic proofreading, known to be essential for the fidelity of protein and DNA synthesis. A variant of this scheme is also described in which a requirement for formation of large aggregates may lead to a further enhancement of the specificity of T-cell activation. Through these mechanisms, ligands of different affinity potentially may elicit qualitatively different signals.

Localization of the Chinese hamster MHC locus to chromosome 1q17-->q18 by fluorescence in situ hybridization.

Comparative gene mapping analysis in mammals suggests that the major histocompatibility complex (MHC) genes map syntenic with genes, such as glyoxylase 1 (GLO1). In man, the MHC locus and other genes of this syntenic group map to chromosome band 6p21.3, and in mouse, these genes map to chromosome 17. In the hamster, however, only the GLO1 gene has been localized; GLO1 maps to chromosome 1, suggesting that the genes within the MHC locus also map to this chromosome. We have localized the hamster MHC class I genes to chromosome band 1q17-->q18 by fluorescence in situ hybridization (FISH). These results suggest that GLO1 and other syntenic genes also lie within this chromosome region.

Genomic structure of the candidate proto-oncogene BCL3.

We previously reported the identification of a novel candidate proto-oncogene involved in the translocation t(14;19)(q32;q13) found in some cases of human B-cell chronic lymphocytic leukemia. This gene, BCL3, is a member of the I kappa B family, whose encoded proteins regulate the NF-kappa B family of transcription factors. Here we describe the genomic structure of BCL3. The gene contains nine exons, spanning 11.5 kb. In comparison to other members of the I kappa B family, there is a remarkable conservation of the exon-intron boundaries in relation to the coding sequences, consistent with an origin from a common ancestral gene. BCL3 is unusual in containing two CpG islands, a 5' island encompassing the first exon, the other lying within the gene. Southern blot analysis using methylation-sensitive restriction enzymes revealed that while the 5' CpG is unmethylated in all tissues tested, the degree of methylation of the internal CpG island varies.

BCL3 encodes a nuclear protein which can alter the subcellular location of NF-kappa B proteins.

BCL3 is a candidate proto-oncogene involved in the recurring translocation t(14;19) found in some patients with chronic lymphocytic leukemia. BCL3 protein acts as an I kappa B in that it can specifically inhibit the DNA binding of NF-kappa B factors. Here, we demonstrate that BCL3 is predominantly a nuclear protein and provide evidence that its N terminus is necessary to direct the protein into the nucleus. In contrast to I kappa B alpha (MAD3), BCL3 does not cause NF-kappa B p50 to be retained in the cytoplasm; instead, in cotransfection assays, it alters the subnuclear localization of p50. The two proteins colocalize, suggesting that they interact in vivo. Further immunofluorescence experiments showed that a mutant p50, lacking a nuclear localization signal and restricted to the cytoplasm, is brought into the nucleus in the presence of BCL3. Correspondingly, a wild-type p50 directs into the nucleus a truncated BCL3, which, when transfected alone, is found in the cytoplasm. We tested whether BCL3 could overcome the cytoplasmic retention of p50 by I kappa B alpha. Results from triple cotransfection experiments with BCL3, I kappa B alpha, and p50 implied that BCL3 can successfully compete with I kappa B alpha and bring p50 into the nucleus; thus, localization of NF-kappa B factors may be affected by differential expression of I kappa B proteins. These novel properties of BCL3 protein further establish BCL3 as a distinctive member of the I kappa B family.