Publications by authors named "Erica Ballabio"

28 Publications

  • Page 1 of 1

Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation.

Cell Rep 2021 May;35(6):109101

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.

Depleting the microenvironment of important nutrients such as arginine is a key strategy for immune evasion by cancer cells. Many tumors overexpress arginase, but it is unclear how these cancers, but not T cells, tolerate arginine depletion. In this study, we show that tumor cells synthesize arginine from citrulline by upregulating argininosuccinate synthetase 1 (ASS1). Under arginine starvation, ASS1 transcription is induced by ATF4 and CEBPβ binding to an enhancer within ASS1. T cells cannot induce ASS1, despite the presence of active ATF4 and CEBPβ, as the gene is repressed. Arginine starvation drives global chromatin compaction and repressive histone methylation, which disrupts ATF4/CEBPβ binding and target gene transcription. We find that T cell activation is impaired in arginine-depleted conditions, with significant metabolic perturbation linked to incomplete chromatin remodeling and misregulation of key genes. Our results highlight a T cell behavior mediated by nutritional stress, exploited by cancer cells to enable pathological immune evasion.
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http://dx.doi.org/10.1016/j.celrep.2021.109101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131582PMC
May 2021

BET inhibition disrupts transcription but retains enhancer-promoter contact.

Nat Commun 2021 01 11;12(1):223. Epub 2021 Jan 11.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, NIHR Oxford Biomedical Research Centre Haematology Theme, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Enhancers are DNA sequences that enable complex temporal and tissue-specific regulation of genes in higher eukaryotes. Although it is not entirely clear how enhancer-promoter interactions can increase gene expression, this proximity has been observed in multiple systems at multiple loci and is thought to be essential for the maintenance of gene expression. Bromodomain and Extra-Terminal domain (BET) and Mediator proteins have been shown capable of forming phase condensates and are thought to be essential for super-enhancer function. Here, we show that targeting of cells with inhibitors of BET proteins or pharmacological degradation of BET protein Bromodomain-containing protein 4 (BRD4) has a strong impact on transcription but very little impact on enhancer-promoter interactions. Dissolving phase condensates reduces BRD4 and Mediator binding at enhancers and can also strongly affect gene transcription, without disrupting enhancer-promoter interactions. These results suggest that activation of transcription and maintenance of enhancer-promoter interactions are separable events. Our findings further indicate that enhancer-promoter interactions are not dependent on high levels of BRD4 and Mediator, and are likely maintained by a complex set of factors including additional activator complexes and, at some sites, CTCF and cohesin.
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http://dx.doi.org/10.1038/s41467-020-20400-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801379PMC
January 2021

Rationale for targeting BCL6 in -rearranged acute lymphoblastic leukemia.

Genes Dev 2019 09 8;33(17-18):1265-1279. Epub 2019 Aug 8.

Department of Systems Biology, City of Hope Comprehensive Cancer Center, Monrovia, California 91016, USA.

Chromosomal rearrangements of the () gene occur in ∼10% of B-cell acute lymphoblastic leukemia (B-ALL) and define a group of patients with dismal outcomes. Immunohistochemical staining of bone marrow biopsies from most of these patients revealed aberrant expression of BCL6, a transcription factor that promotes oncogenic B-cell transformation and drug resistance in B-ALL. Our genetic and ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analyses showed that MLL-AF4 and MLL-ENL fusions directly bound to the promoter and up-regulated BCL6 expression. While oncogenic MLL fusions strongly induced aberrant BCL6 expression in B-ALL cells, germline was required to up-regulate Bcl6 in response to physiological stimuli during normal B-cell development. Inducible expression of Bcl6 increased mRNA levels, which was reversed by genetic deletion and pharmacological inhibition of Bcl6, suggesting a positive feedback loop between MLL and BCL6. Highlighting the central role of BCL6 in rearranged B-ALL, conditional deletion and pharmacological inhibition of BCL6 compromised leukemogenesis in transplant recipient mice and restored sensitivity to vincristine chemotherapy in rearranged B-ALL patient samples. Oncogenic MLL fusions strongly induced transcriptional activation of the proapoptotic BH3-only molecule BIM, while BCL6 was required to curb MLL-induced expression of BIM. Notably, peptide (RI-BPI) and small molecule (FX1) BCL6 inhibitors derepressed BIM and synergized with the BH3-mimetic ABT-199 in eradicating rearranged B-ALL cells. These findings uncover MLL-dependent transcriptional activation of BCL6 as a previously unrecognized requirement of malignant transformation by oncogenic MLL fusions and identified BCL6 as a novel target for the treatment of MLL-rearranged B-ALL.
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http://dx.doi.org/10.1101/gad.327593.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719625PMC
September 2019

The basic helix-loop-helix transcription factor SHARP1 is an oncogenic driver in MLL-AF6 acute myelogenous leukemia.

Nat Commun 2018 04 24;9(1):1622. Epub 2018 Apr 24.

Cancer Science Institute of Singapore, National University of Singapore, Singapore, 117599, Singapore.

Acute Myeloid Leukemia (AML) with MLL gene rearrangements demonstrate unique gene expression profiles driven by MLL-fusion proteins. Here, we identify the circadian clock transcription factor SHARP1 as a novel oncogenic target in MLL-AF6 AML, which has the worst prognosis among all subtypes of MLL-rearranged AMLs. SHARP1 is expressed solely in MLL-AF6 AML, and its expression is regulated directly by MLL-AF6/DOT1L. Suppression of SHARP1 induces robust apoptosis of human MLL-AF6 AML cells. Genetic deletion in mice delays the development of leukemia and attenuated leukemia-initiating potential, while sparing normal hematopoiesis. Mechanistically, SHARP1 binds to transcriptionally active chromatin across the genome and activates genes critical for cell survival as well as key oncogenic targets of MLL-AF6. Our findings demonstrate the unique oncogenic role for SHARP1 in MLL-AF6 AML.
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http://dx.doi.org/10.1038/s41467-018-03854-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915391PMC
April 2018

MLL-AF4 Spreading Identifies Binding Sites that Are Distinct from Super-Enhancers and that Govern Sensitivity to DOT1L Inhibition in Leukemia.

Cell Rep 2017 01;18(2):482-495

MRC, Molecular Haematology Unit, NIHR Oxford Biomedical Research Centre Programme, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK. Electronic address:

Understanding the underlying molecular mechanisms of defined cancers is crucial for effective personalized therapies. Translocations of the mixed-lineage leukemia (MLL) gene produce fusion proteins such as MLL-AF4 that disrupt epigenetic pathways and cause poor-prognosis leukemias. Here, we find that at a subset of gene targets, MLL-AF4 binding spreads into the gene body and is associated with the spreading of Menin binding, increased transcription, increased H3K79 methylation (H3K79me2/3), a disruption of normal H3K36me3 patterns, and unmethylated CpG regions in the gene body. Compared to other H3K79me2/3 marked genes, MLL-AF4 spreading gene expression is downregulated by inhibitors of the H3K79 methyltransferase DOT1L. This sensitivity mediates synergistic interactions with additional targeted drug treatments. Therefore, epigenetic spreading and enhanced susceptibility to epidrugs provides a potential marker for better understanding combination therapies in humans.
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http://dx.doi.org/10.1016/j.celrep.2016.12.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5263239PMC
January 2017

MLL-AF4 binds directly to a BCL-2 specific enhancer and modulates H3K27 acetylation.

Exp Hematol 2017 03 14;47:64-75. Epub 2016 Nov 14.

Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, University of Oxford, Headington, Oxford, UK. Electronic address:

Survival rates for children and adults carrying mutations in the Mixed Lineage Leukemia (MLL) gene continue to have a very poor prognosis. The most common MLL mutation in acute lymphoblastic leukemia is the t(4;11)(q21;q23) chromosome translocation that fuses MLL in-frame with the AF4 gene producing MLL-AF4 and AF4-MLL fusion proteins. Previously, we found that MLL-AF4 binds to the BCL-2 gene and directly activates it through DOT1L recruitment and increased H3K79me2/3 levels. In the study described here, we performed a detailed analysis of MLL-AF4 regulation of the entire BCL-2 family. By measuring nascent RNA production in MLL-AF4 knockdowns, we found that of all the BCL-2 family genes, MLL-AF4 directly controls the active transcription of both BCL-2 and MCL-1 and also represses BIM via binding of the polycomb group repressor 1 (PRC1) complex component CBX8. We further analyzed MLL-AF4 activation of the BCL-2 gene using Capture-C and identified a BCL-2-specific enhancer, consisting of two clusters of H3K27Ac at the 3' end of the gene. Loss of MLL-AF4 activity results in a reduction of H3K79me3 levels in the gene body and H3K27Ac levels at the 3' BCL-2 enhancer, revealing a novel regulatory link between these two histone marks and MLL-AF4-mediated activation of BCL-2.
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http://dx.doi.org/10.1016/j.exphem.2016.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333536PMC
March 2017

MLL-Rearranged Acute Lymphoblastic Leukemias Activate BCL-2 through H3K79 Methylation and Are Sensitive to the BCL-2-Specific Antagonist ABT-199.

Cell Rep 2015 Dec 17;13(12):2715-27. Epub 2015 Dec 17.

Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias.
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http://dx.doi.org/10.1016/j.celrep.2015.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700051PMC
December 2015

Self-enforcing feedback activation between BCL6 and pre-B cell receptor signaling defines a distinct subtype of acute lymphoblastic leukemia.

Cancer Cell 2015 Mar;27(3):409-25

Division of Pediatric Hematology and Oncology, Department of Pediatrics, Oregon Health & Science University, Portland, OR 97239, USA; Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR 97239, USA.

Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR(+) ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR(+) ALL.
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http://dx.doi.org/10.1016/j.ccell.2015.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618684PMC
March 2015

Epigenetic control of gene expression in leukemogenesis: Cooperation between wild type MLL and MLL fusion proteins.

Mol Cell Oncol 2014 Apr-Jun;1(2):e955330. Epub 2014 Oct 29.

MRC Molecular Hematology Unit; Weatherall Institute of Molecular Medicine; University of Oxford ; Oxford, UK.

Although there has been great progress in the treatment of human cancers, especially leukemias, many remain resistant to treatment. A major current focus is the development of so-called epigenetic drugs. Epigenetic states are stable enough to persist through multiple cell divisions, but by their very nature are reversible and thus are amenable to therapeutic manipulation. Exciting work in this area has produced a new breed of highly specific small molecules designed to inhibit epigenetic proteins, some of which have entered clinical trials. The current and future development of epigenetic drugs is greatly aided by highly detailed information about normal and aberrant epigenetic changes at the molecular level. In this review we focus on a class of aggressive acute leukemias caused by mutations in the Mixed Lineage Leukemia (MLL) gene. We provide an overview of how detailed molecular analysis of MLL leukemias has provided several early-stage epigenetic drugs and propose that further study of MLL leukemogenesis may continue to provide molecular details that potentially have a wider range of applications in human cancers.
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http://dx.doi.org/10.1080/23723548.2014.955330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905190PMC
June 2016

RUNX1 is a key target in t(4;11) leukemias that contributes to gene activation through an AF4-MLL complex interaction.

Cell Rep 2013 Jan 24;3(1):116-27. Epub 2013 Jan 24.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.

The Mixed Lineage Leukemia (MLL) protein is an important epigenetic regulator required for the maintenance of gene activation during development. MLL chromosomal translocations produce novel fusion proteins that cause aggressive leukemias in humans. Individual MLL fusion proteins have distinct leukemic phenotypes even when expressed in the same cell type, but how this distinction is delineated on a molecular level is poorly understood. Here, we highlight a unique molecular mechanism whereby the RUNX1 gene is directly activated by MLL-AF4 and the RUNX1 protein interacts with the product of the reciprocal AF4-MLL translocation. These results support a mechanism of transformation whereby two oncogenic fusion proteins cooperate by activating a target gene and then modulating the function of its downstream product.
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http://dx.doi.org/10.1016/j.celrep.2012.12.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3607232PMC
January 2013

Primary cutaneous anaplastic large cell lymphoma shows a distinct miRNA expression profile and reveals differences from tumor-stage mycosis fungoides.

Exp Dermatol 2012 Aug;21(8):632-4

Department of Dermatology, LUMC, Leiden, The Netherlands.

The miRNA expression profiles of skin biopsies from 14 primary cutaneous anaplastic large cell lymphoma (C-ALCL) patients were analysed with miRNA microarrays using the same control group of 12 benign inflammatory dermatoses (BID) as previously used to study the miRNA expression profile of tumor-stage mycosis fungoides (MF). We identified 13 differentially expressed miRNAs between C-ALCL and BID. The up-regulation of miR-155, miR-27b, miR-30c and miR-29b in C-ALCL was validated by miRNA-Q-PCR on independent study groups. Additionally, the miRNA expression profiles of C-ALCL were compared with those of tumor-stage MF. Although miRNA microarray analysis did not identify statistically significant differentially expressed miRNAs, miRNA-Q-PCR demonstrated statistically significantly differential expression of miR-155, miR-27b, miR-93, miR-29b and miR-92a between tumor-stage MF and C-ALCL. This study, the first describing the miRNA expression profile of C-ALCL, reveals differences with tumor-stage MF, suggesting a different contribution to the pathogenesis of these lymphomas.
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http://dx.doi.org/10.1111/j.1600-0625.2012.01548.xDOI Listing
August 2012

Inter- and intra-observational variability in immunohistochemistry: a multicentre analysis of diffuse large B-cell lymphoma staining.

Histopathology 2012 Jul 28;61(1):18-25. Epub 2012 Feb 28.

Biodonostia Research Institute, San Sebastián, and IKERBASQUE, Basque Foundation for Science, Bilbao, Spain.

Aims: Although many immunohistochemical (IHC) cancer biomarkers have been identified, very few have translated into routine clinical practice, primarily because of technical and observational inconsistencies between studies. However, despite the obvious need to address such variability, very few studies have done so.

Methods And Results: Using bcl-6, CD10, MUM1, GCET1 and FOXP1 antibody staining on diffuse large B-cell lymphoma cases (n = 138) as a model, we employed Cronbach α analysis to quantify interobserver and intraobserver variability between four independent observers (two per institution), scoring two tissue microarrays (TMAs) stained at both institutions using differing staining procedures. The overall concordance between all observations irrespective of staining procedure or TMA source was high (average α = 0.951), with the highest level being reached for CD10 staining (average α = 0.967) and the lowest for bcl-6 (average α = 0.924). Interslide and interinstitutional reproducibility were similarly high (average α = 0.952 and average α = 0.934, respectively). Interobserver/intrainstitutional and interobserver/interinstitutional comparisons showed lower levels of concordance (average α = 0.870 and average α = 0.877, respectively), and intraobserver/interinstitutional comparisons showed the lowest levels of concordance (average α = 0.810), particularly for bcl-6 staining (α = 0.658).

Conclusions: This study suggests that most variability in IHC studies between centres results from inherent limitations of the biomarkers investigated rather than procedural or observational differences.
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http://dx.doi.org/10.1111/j.1365-2559.2012.04179.xDOI Listing
July 2012

Molecular and Epigenetic Mechanisms of MLL in Human Leukemogenesis.

Cancers (Basel) 2012 Sep 10;4(3):904-44. Epub 2012 Sep 10.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital Headington, Oxford OX3 9DS, UK.

Epigenetics is often defined as the study of heritable changes in gene expression or chromosome stability that don't alter the underlying DNA sequence. Epigenetic changes are established through multiple mechanisms that include DNA methylation, non-coding RNAs and the covalent modification of specific residues on histone proteins. It is becoming clear not only that aberrant epigenetic changes are common in many human diseases such as leukemia, but that these changes by their very nature are malleable, and thus are amenable to treatment. Epigenetic based therapies have so far focused on the use of histone deacetylase (HDAC) inhibitors and DNA methyltransferase inhibitors, which tend to have more general and widespread effects on gene regulation in the cell. However, if a unique molecular pathway can be identified, diseases caused by epigenetic mechanisms are excellent candidates for the development of more targeted therapies that focus on specific gene targets, individual binding domains, or specific enzymatic activities. Designing effective targeted therapies depends on a clear understanding of the role of epigenetic mutations during disease progression. The Mixed Lineage Leukemia (MLL) protein is an example of a developmentally important protein that controls the epigenetic activation of gene targets in part by methylating histone 3 on lysine 4. MLL is required for normal development, but is also mutated in a subset of aggressive human leukemias and thus provides a useful model for studying the link between epigenetic cell memory and human disease. The most common MLL mutations are chromosome translocations that fuse the MLL gene in frame with partner genes creating novel fusion proteins. In this review, we summarize recent work that argues MLL fusion proteins could function through a single molecular pathway, but we also highlight important data that suggests instead that multiple independent mechanisms underlie MLL mediated leukemogenesis.
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http://dx.doi.org/10.3390/cancers4030904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712720PMC
September 2012

Genomic imbalances are confined to non-proliferating cells in paediatric patients with acute myeloid leukaemia and a normal or incomplete karyotype.

PLoS One 2011 9;6(6):e20607. Epub 2011 Jun 9.

Centre for Cell and Chromosome Biology and Brunel Institute of Cancer Genetics and Pharmacogenomics, Division of Biosciences, Brunel University, West London, United Kingdom.

Leukaemia is often associated with genetic alterations such as translocations, amplifications and deletions, and recurrent chromosome abnormalities are used as markers of diagnostic and prognostic relevance. However, a proportion of acute myeloid leukaemia (AML) cases have an apparently normal karyotype despite comprehensive cytogenetic analysis. Based on conventional cytogenetic analysis of banded chromosomes, we selected a series of 23 paediatric patients with acute myeloid leukaemia and performed whole genome array comparative genome hybridization (aCGH) using DNA samples derived from the same patients. Imbalances involving large chromosomal regions or entire chromosomes were detected by aCGH in seven of the patients studied. Results were validated by fluorescence in situ hybridization (FISH) to both interphase nuclei and metaphase chromosomes using appropriate bacterial artificial chromosome (BAC) probes. The majority of these copy number alterations (CNAs) were confirmed by FISH and found to localize to the interphase rather than metaphase nuclei. Furthermore, the proliferative states of the cells analyzed by FISH were tested by immunofluorescence using an antibody against the proliferation marker pKi67. Interestingly, these experiments showed that, in the vast majority of cases, the changes appeared to be confined to interphase nuclei in a non-proliferative status.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0020607PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111408PMC
November 2011

MicroRNA expression in multiple myeloma is associated with genetic subtype, isotype and survival.

Biol Direct 2011 May 18;6:23. Epub 2011 May 18.

Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, UK.

Background: MicroRNAs are small RNA species that regulate gene expression post-transcriptionally and are aberrantly expressed in many cancers including hematological malignancies. However, the role of microRNAs in the pathogenesis of multiple myeloma (MM) is only poorly understood. We therefore used microarray analysis to elucidate the complete miRNome (miRBase version 13.0) of purified tumor (CD138+) cells from 33 patients with MM, 5 patients with monoclonal gammopathy of undetermined significance (MGUS) and 9 controls.

Results: Unsupervised cluster analysis revealed that MM and MGUS samples have a distinct microRNA expression profile from control CD138+ cells. The majority of microRNAs aberrantly expressed in MM (109/129) were up-regulated. A comparison of these microRNAs with those aberrantly expressed in other B-cell and T-cell malignancies revealed a surprising degree of similarity (~40%) suggesting the existence of a common lymphoma microRNA signature. We identified 39 microRNAs associated with the pre-malignant condition MGUS. Twenty-three (59%) of these were also aberrantly expressed in MM suggesting common microRNA expression events in MM progression. MM is characterized by multiple chromosomal abnormalities of varying prognostic significance. We identified specific microRNA signatures associated with the most common IgH translocations (t(4;14) and t(11;14)) and del(13q). Expression levels of these microRNAs were distinct between the genetic subtypes (by cluster analysis) and correctly predicted these abnormalities in > 85% of cases using the support vector machine algorithm. Additionally, we identified microRNAs associated with light chain only myeloma, as well as IgG and IgA-type MM. Finally, we identified 32 microRNAs associated with event-free survival (EFS) in MM, ten of which were significant by univariate (logrank) survival analysis.

Conclusions: In summary, this work has identified aberrantly expressed microRNAs associated with the diagnosis, pathogenesis and prognosis of MM, data which will prove an invaluable resource for understanding the role of microRNAs in this devastating disease.
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http://dx.doi.org/10.1186/1745-6150-6-23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3120802PMC
May 2011

miRNA expression profiling of mycosis fungoides.

Mol Oncol 2011 Jun 24;5(3):273-80. Epub 2011 Feb 24.

Department of Dermatology, LUMC, Leiden, The Netherlands.

MicroRNAs (miRNAs) are small RNA species that regulate gene expression post-transcriptionally and are aberrantly expressed in many malignancies including lymphoma. However, the role of miRNAs in the pathogenesis of T-cell lymphoid malignancies is poorly understood. Previously we examined the miRNA profile of Sézary syndrome (Sz), a leukemia of skin-homing memory T cells. In this study we determined the complete miRNome of mycosis fungoides (MF), the most common type of cutaneous T cell lymphoma. The miRNA profile of skin biopsies from 19 patients with tumor stage MF and 12 patients with benign inflammatory dermatoses (eczema and lichen planus) were compared by microarray analysis. We identified 49 miRNAs that are differentially expressed in tumor stage MF compared to benign inflammatory dermatoses using ANOVA analysis (P < 0.05, Benjamini-Hochberg corrected). The majority of the differentially expressed miRNAs (30/49) were up-regulated in tumor stage MF. The most significant differentially expressed were miR-155 and miR-92a (both up-regulated in tumor stage MF), while miR-93 showed the highest up-regulation in tumor stage MF with a fold difference of 5.8. Differential expression of a selection of these miRNAs was validated by miRNA-Q-PCR on additional test groups (tumors and controls). None of the miRNAs up-regulated in tumor stage MF was previously shown to be up-regulated in Sz, and only 2 of the 19 miRNAs down-regulated in tumor stage MF were also down-regulated in Sz. Taken together this report is the first describing the miRNA signature of tumor stage MF.
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http://dx.doi.org/10.1016/j.molonc.2011.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5528293PMC
June 2011

MicroRNA expression in Sezary syndrome: identification, function, and diagnostic potential.

Blood 2010 Aug 6;116(7):1105-13. Epub 2010 May 6.

Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.

MicroRNAs are commonly aberrantly expressed in many cancers. Very little is known of their role in T-cell lymphoma, however. We therefore elucidated the complete miRNome of purified T cells from 21 patients diagnosed with Sézary Syndrome (SzS), a rare aggressive primary cutaneous T-cell (CD4(+)) lymphoma. Unsupervised cluster analysis of microarray data revealed that the microRNA expression profile was distinct from CD4(+) T-cell controls and B-cell lymphomas. The majority (104 of 114) of SzS-associated microRNAs (P < .05) were down-regulated and their expression pattern was largely consistent with previously reported genomic copy number abnormalities and were found to be highly enriched (P < .001) for aberrantly expressed target genes. Levels of miR-223 distinguished SzS samples (n = 32) from healthy controls (n = 19) and patients with mycosis fungoides (n = 11) in more than 90% of samples. Furthermore, we demonstrate that the down-regulation of intronically encoded miR-342 plays a role in the pathogenesis of SzS by inhibiting apoptosis, and describe a novel mechanism of regulation for this microRNA via binding of miR-199a* to its host gene. We also provide the first in vivo evidence for down-regulation of the miR-17-92 cluster in malignancy and demonstrate that ectopic miR-17-5p expression increases apoptosis and decreases cell proliferation in SzS cells.
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http://dx.doi.org/10.1182/blood-2009-12-256719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2938132PMC
August 2010

The inducible T-cell co-stimulator molecule is expressed on subsets of T cells and is a new marker of lymphomas of T follicular helper cell-derivation.

Haematologica 2010 Mar;95(3):432-9

Leukaemia Research Immunodiagnostics Unit, Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, Oxford, OX3 9DU, UK.

Background: T follicular helper (T(FH)) cells reside in the light zone of germinal centers and are considered the cell of origin of angioimmunoblastic T-cell lymphoma. Recently, CXCL13, PD-1 and SAP were described as useful markers for T(FH) cells and angioimmunoblastic T-cell lymphoma but also reported in some peripheral T-cell lymphomas, not otherwise specified.

Design And Methods: In the present study the expression pattern of ICOS protein was investigated by immunohistochemistry-based techniques in routine sections of normal lymphoid tissues and 633 human lymphomas.

Results: Cells strongly positive for ICOS were restricted to the light zone of germinal centers and co-expressed T(FH)-associated molecules. In addition, weak to moderate ICOS expression was observed in a small proportion of FOXP3-positive cells. In lymphomas, ICOS expression was confined to angioimmunoblastic T-cell lymphoma (85/86), peripheral T-cell lymphomas of follicular variant (18/18) and a proportion of peripheral T-cell lymphomas, not otherwise specified (24/56) that also expressed other T(FH)-associated molecules.

Conclusions: ICOS is a useful molecule for identifying T(FH) cells and its restricted expression to angioimmunoblastic T-cell lymphoma and a proportion of peripheral T-cell lymphomas, not otherwise specified (showing a T(FH)-like profile) suggests its inclusion in the antibody panel for diagnosing T(FH)-derived lymphomas. Our findings provide further evidence that the histological spectrum of T(FH)-derived lymphomas is broader than previously assumed.
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http://dx.doi.org/10.3324/haematol.2009.010991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2833073PMC
March 2010

Expression of microRNAs in diffuse large B cell lymphoma is associated with immunophenotype, survival and transformation from follicular lymphoma.

J Cell Mol Med 2009 Jul 24;13(7):1248-60. Epub 2008 Dec 24.

Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, UK.

MicroRNAs are naturally occurring small RNA species that regulate gene expression and are frequently abnormally expressed in cancers. However, the role of microRNAs in lymphoma is poorly understood. Therefore, we undertook a comprehensive study of microRNA expression in two of the most common lymphomas: diffuse large B-cell lymphoma (DLBCL) (n = 80) and follicular lymphoma (FCL) (n = 18) using microarrays containing probes for 464 human microRNAs. Unsupervised cluster analysis revealed distinct expression patterns between these two lymphomas and specific microRNA signatures (including members of the miR-17-92 cluster) were derived that correctly predicted lymphoma type in >95% of cases. Furthermore, we identified microRNAs in de novo DLBCL (n = 64) associated with germinal centre-like and non-germinal centre-like immunophenotypes, international prognostic index status and event-free survival in CHOP and rituximab (R)-CHOP treated patients. Despite the indolent nature of FCL a significant proportion of cases undergo high-grade transformation to more aggressive DLBCL. In order to see if transformation is associated with changes in microRNA expression we compared transformed DLBCL cases (n = 16) with de novo DLBCL, as well as FCL cases that underwent subsequent transformation (n = 7) with FCL cases that had not transformed at a median follow-up of 60 months (n = 11). Differential expression of 12 microRNAs correctly predicted >85% of transformed versus de novo DLBCL cases; six microRNAs (miR-223, 217, 222, 221 and let-7i and 7b) were found which could similarly predict or transformation in FCL (P < 0.05). These data suggest that microRNAs have potential as diagnostic and prognostic markers in these lymphomas and may be used to identify FCL patients at risk of high-grade transformation.
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http://dx.doi.org/10.1111/j.1582-4934.2008.00628.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496139PMC
July 2009

Aberrant expression of microRNA biosynthetic pathway components is a common feature of haematological malignancy.

Br J Haematol 2009 May 2;145(4):545-8. Epub 2008 Mar 2.

Lymphoid Malignancy Research Group, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, UK.

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http://dx.doi.org/10.1111/j.1365-2141.2009.07642.xDOI Listing
May 2009

Labeling of multiple cell markers and mRNA using automated apparatus.

Appl Immunohistochem Mol Morphol 2008 Jul;16(4):371-81

Leukaemia Research Immunodiagnostics Unit, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, UK.

Double immunoenzymatic labeling of 2 different molecules in tissue sections is a widely used technique. However, it is time consuming since the 2 immunoenzymatic procedures are carried out in sequence, and they must also be optimally performed to avoid unwanted background labeling. In this paper, we report that double immunoenzymatic staining performed using automated immunostaining apparatus considerably reduces the requirements in terms of time and is also highly reproducible and free of background. Three tissue markers can also be visualized by performing (after immunoperoxidase labeling) 2 sequential immuno-alkaline phosphatase procedures using different substrates. Furthermore, single or double detection of mRNA by in situ hybridization can be combined with immunoenzymatic labeling. Finally, automated labeling could also be performed on peripheral blood and bone marrow smears, opening the possibility of using this procedure in the analysis of hematologic/cytology samples.
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http://dx.doi.org/10.1097/PAI.0b013e318164fc63DOI Listing
July 2008

Novel markers of normal and neoplastic human plasmacytoid dendritic cells.

Blood 2008 Apr 24;111(7):3778-92. Epub 2008 Jan 24.

Leukaemia Research Fund Immunodiagnostics Unit, Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, Oxford, UK.

Plasmacytoid dendritic cells (pDCs) are involved in innate immunity (eg, by secreting interferons) and also give rise to CD4+CD56+ hematodermic neoplasms. We report extensive characterization of human pDCs in routine tissue samples, documenting the expression of 19 immunohistologic markers, including signaling molecules (eg, BLNK), transcription factors (eg, ICSBP/IRF8 and PU.1), and Toll-like receptors (TLR7, TLR9). Many of these molecules are expressed in other cell types (principally B cells), but the adaptor protein CD2AP was essentially restricted to pDCs, and is therefore a novel immunohistologic marker for use in tissue biopsies. We found little evidence for activation-associated morphologic or phenotypic changes in conditions where pDCs are greatly increased (eg, Kikuchi disease). Most of the molecules were retained in the majority of pDC neoplasms, and 3 (BCL11A, CD2AP, and ICSBP/IRF8) were also commonly negative in leukemia cutis (acute myeloid leukemia in the skin), a tumor that may mimic pDC neoplasia. In summary, we have documented a range of molecules (notably those associated with B cells) expressed by pDCs in tissues and peripheral blood (where pDCs were detectable in cytospins at a frequency of <1% of mononuclear cells) and also defined potential new markers (in particular CD2AP) for the diagnosis of pDC tumors.
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http://dx.doi.org/10.1182/blood-2007-10-117531DOI Listing
April 2008

Expression of two markers of germinal center T cells (SAP and PD-1) in angioimmunoblastic T-cell lymphoma.

Haematologica 2007 Aug 20;92(8):1059-66. Epub 2007 Jul 20.

Monoclonal Antibodies Unit, Biotechnology Program,Spanish National Cancer Centre, Madrid, Spain.

Background And Objectives: In the present paper we report that SAP, an intracytoplasmic molecule that is involved in cell signaling, is an immunohistologic marker for germinal center T cells in paraffin-embedded tissue. We document its expression, and also that of PD-1 (another recently described marker of germinal center T cells to which a new antibody has been raised), in normal and neoplastic lymphoid tissue to evaluate the suggestion that helper T cells within the germinal centers of human lymphoid tissue are the cell of origin of angioimmunoblastic T-cell lymphoma (AITL), and to assess the diagnostic value of these two markers.

Design And Methods: Expression of SAP and PD-1 was investigated by immunohistochemistry in paraffin-embedded tissue sections and in cell lines. Western blotting was performed on cell lines, and antibody specificity was confirmed by immunostaining of transfected cells. RESULTS Screening on more than 500 lymphoma biopsies showed that 95% (40/42) of cases of AITL expressed at least one of these markers. SAP was also expressed on many cases (15/21) of acute T lymphoblastic leukemia, in keeping with its presence in cortical thymocytes. However, PD-1 and SAP were also found in a minority of cases of peripheral T-cell lymphoma other than AITL, in contrast to a report that the former marker is specific for AITL. This observation raises the possibility that such non-angioimmunoblastic cases may be related to germinal center helper T cells.

Interpretation And Conclusions: These two markers provide additional evidence that AITL arises from germinal center T cells. They may also prove of value in the diagnosis of this disease since a negative reaction was rarely observed in this disorder.
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http://dx.doi.org/10.3324/haematol.10864DOI Listing
August 2007

Differential expression of EDF-1 and endothelial nitric oxide synthase by proliferating, quiescent and senescent microvascular endothelial cells.

Biochim Biophys Acta 2005 Sep;1745(2):265-72

University of Milan, Department of Preclinical Sciences LITA Vialba, Via GB Grassi, 74 20157 Milan, Italy.

Endothelial Differentiation-related Factor (EDF)-1 is a low molecular weight polypeptide downregulated in endothelial cells exposed to HIV-1-Tat or the phorbol ester TPA. EDF-1 acts in the cytosol as a calmodulin binding protein, and in the nucleus as a transcriptional coactivator. Here, we show that EDF-1 is downregulated in non-proliferating microvascular endothelial cells. Indeed, both quiescence and senescence reduce the levels of EDF-1 and this is due to protein degradation through the proteasome. We also describe a different subcellular localization of EDF-1 which is mainly nuclear in senescent 1G11 cells. Since (i) endothelial nitric oxide (NO) seems to play a role in endothelial proliferation and (ii) NO is an important mediator involved in the control of vascular tone, inflammatory responses and angiogenesis, it is noteworthy that senescence downregulates the expression and the activity of endothelial nitric oxide synthase (eNOS) in microvascular endothelial cells. On the contrary, quiescence does not affect NOS expression and activity. The modulation of EDF-1 in microvascular endothelial cells might offer new insights into the molecular events involved in angiogenesis and in microvascular dysfunctions in the elderly.
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http://dx.doi.org/10.1016/j.bbamcr.2005.06.013DOI Listing
September 2005

Characterization of 6q abnormalities in childhood acute myeloid leukemia and identification of a novel t(6;11)(q24.1;p15.5) resulting in a NUP98-C6orf80 fusion in a case of acute megakaryoblastic leukemia.

Genes Chromosomes Cancer 2005 Nov;44(3):225-32

Leukaemia Research Fund Molecular Haematology Unit, Nuffield Department of Clinical Laboratory Science, John Radcliffe Hospital, Oxford, United Kingdom.

Chromosome abnormalities of 6q are not frequently observed in myeloid disorders. In this article, we report the incidence of these chromosome changes in childhood myeloid leukemia as 2%-4% based on the cytogenetic database of a single institution. We applied fluorescence in situ hybridization (FISH) to characterize precisely the types of 6q abnormalities in seven patients (six with acute myeloid leukemia and one with myelodysplastic syndrome). They carried various translocations involving different breakpoints in 6q, as confirmed by FISH using a whole-chromosome-6 paint. Four cases were reported as t(6;11), although the breakpoints varied. Among these, we identified a novel translocation, t(6;11)(q24.1;p15.5), in a patient with acute megakaryoblastic leukemia. Molecular cytogenetic studies using the PAC clone RP5-1173K1 localized the genomic breakpoint on chromosome 11 to within the NUP98 gene. The breakpoint on chromosome 6 was narrowed down to a 500-kb region between BAC clones RP11-721P14 and RP11-39H10. Reverse-transcription PCR was performed using a forward primer specific for NUP98 and a reverse primer for the candidate gene in the 500-kb interval in 6q. This experiment resulted in the identification of a new fusion between NUP98 and C6orf80. Further studies will aim to fully characterize C6orf80 and will elucidate the role of this new NUP98 fusion in myeloid leukemia.
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http://dx.doi.org/10.1002/gcc.20233DOI Listing
November 2005

Endothelial stress by gravitational unloading: effects on cell growth and cytoskeletal organization.

Biochim Biophys Acta 2003 Oct;1642(3):173-9

Department of Preclinical Sciences, LITA Vialba, Università di Milano, Via GB Grassi 74, Milan, Italy.

All organisms on Earth have evolved to survive within the pull of gravity. Orbital space flights have clearly demonstrated that the absence or the reduction of gravity profoundly affects eukaryotic organisms, including man. Because (i). endothelial cells are crucial in the maintenance of the functional integrity of the vascular wall, and (ii). cardiovascular deconditioning has been described in astronauts, we evaluated whether microgravity affected endothelial functions. We show that microgravity reversibly stimulated endothelial cell growth. This effect correlated with an overexpression of heat shock protein 70 (hsp70) and a down-regulation of interleukin 1 alpha (IL-1alpha), a potent inhibitor of endothelial cell growth, also implicated in promoting senescence. In addition, gravitationally unloaded endothelial cells rapidly remodelled their cytoskeleton and, after a few days, markedly down-regulated actin through a transcriptional mechanism. We hypothesize that the reduction in the amounts of actin in response to microgravity represents an adaptative mechanism to avoid the accumulation of redundant actin fibers.
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http://dx.doi.org/10.1016/j.bbamcr.2003.08.003DOI Listing
October 2003