Academic literature on the topic 'Gene di fusione'

Abstract Acute Myeloid Leukemia (AML) is a highly heterogeneous disease and a complex network of events contribute to its pathogenesis. Chromosomal rearrangements and fusion genes have a crucial diagnostic, prognostic and therapeutic role in AML. A recent RNA sequencing (RNAseq) study on 179 AML revealed that fusion events occur in 45% of patients. However, the leukemogenic potential of these fusions and their prognostic role are still unknown. To identify novel rare gene fusions having a causative role in leukemogenesis and to identify potential targets for personalized therapies, transcriptome profiling was performed on AML cases with rare and poorly described chromosomal translocations. Bone marrow samples were collected from 5 AML patients (#59810, #20 and #84 at diagnosis and #21 and #32 at relapse). RNAseq was performed using the Illumina Hiseq2000 platform. The presence of gene fusions was assessed with deFuse and Chimerascan. Putative fusion genes were prioritized using Pegasus and Oncofuse, in order to select biologically relevant fusions. Chimeras not supported by split reads, occurring in reactive samples, involving not annotated or conjoined genes were removed. The remaining fusions were prioritized according to mapping of partner genes to chromosomes involved in the translocation or to Chimerascan and deFuse concordance. The CBFβ-MYH11 chimera was identified in sample #84, carrying inv(16) aberration, thus confirming the reliability of our analysis. Sample #59810 carried the fusion transcript ZEB2-BCL11B (Driver Score, DS=0.7), which is an in-frame fusion and a rare event in AML associated with t(2;14)(q21;q32). The breakpoint of the fusion mapped in exon 2 of ZEB2 (ENST00000558170) and exon 2 of BCL11B (ENST00000357195). Differently from previous data, this fusion transcript showed 3 splicing isoforms. Type 1 isoform is the full-length chimera and it retains all exons of both genes involved in the translocation. Type 2 isoform was characterized by the junction of exon 2 of ZEB2 and exon 3 of BCL11B. In type 3 isoform, exon 2 and 3 of BCL11B were removed, resulting in an mRNA composed by exon 2 of ZEB2 and exon 4 of BCL11B. Gene expression profiling showed an upregulation of ZEB2 and BCL11B transcripts in the patient's blasts, compared to 53 AML samples with no chromosomal aberrations in the 14q32 region. The same samples showed the WT1-CNOT2 chimera, which is a novel out-of-frame fusion (DS= 0.008) related to t(11;12) translocation, identified by cytogenetic analysis. Two new in-frame fusion genes were identified in sample #20: CPD-PXT1 (DS=0.07), which appeared as the reciprocal fusion product of t(6;17) translocation, and SAV1-GYPB, which remained cryptic at cytogenetic analysis (DS=0.8, alternative splicing events are being investigated). SAV1 was downregulated in sample #20 compared to our AML cohort, suggesting the putative loss of a tumour-suppressor gene. Sample #21 carried a t(3;12) translocation and RNAseq identified a novel fusion event between chromosomes 19 and 7, involving the genes OAZ and MAFK (DS=0.9). Finally, no chimeras were confirmed in sample #32 having a t(12;18) translocation. Our data suggest that fusion events are frequent in AML and a number of them cannot be detected by current cytogenetic analyses. Gene fusions cooperate to AML pathogenesis and heterogeneity and we are further investigating the oncogenic potential of the identified translocations. Moreover, the results firmly indicate that different approaches, including G-banding, molecular biology, bioinformatics and statistics, need to be integrated in order to better understand AML pathogenesis and improve patients' stratification, High-resolution sequencing analysis currently represent the most informative strategy to tailor personalized therapies. Acknowledgments: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:BMS: Speakers Bureau; MSD: Consultancy; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

Operons are rare in eukaryotes, where they often allow concerted expression of functionally related genes. While a dicistronic transcription unit encoding two unrelated genes, the suppressor of position-effect variegation su(var)3-9 and the gamma subunit of eukaryotic translation initiation factor 2 (eIF2γ) has been found in insecta, and its significance is not well understood. Here, we analyzed the evolutionary history of this transcription unit in arthropods and its functions by using model Coleoptera insect Tribolium castaneum. In T. castaneum, Tcsu(var)3-9 fused into the 80 N-terminal amino acids of TceIF2γ, the transcription of these two genes are resolved by alternative splicing. Phylogenetic analysis supports the natural gene fusion of su(var)3-9 and eIF2γ occurred in the ancestral line of winged insects and silverfish, but with frequent re-fission during the evolution of insects. Functional analysis by using RNAi for these two genes revealed that gene fusion did not invoke novel functions for the gene products. As a histone methyltransferase, Tcsu(var)3-9 is primarily responsible for H3K9 di-, and tri-methylation and plays important roles in metamorphosis and embryogenesis in T. castaneum. While TceIF2γ plays essential roles in T. castaneum by positively regulating protein translation mediated ecdysteroid biosynthesis. The vulnerability of the gene fusion and totally different role of su(var)3-9 and eIF2γ in T. castaneum confirm this gene fusion is a non-selected, constructive neutral evolution event in insect. Moreover, the positive relationship between protein translation and ecdysteroid biosynthesis gives new insights into correlations between translation regulation and hormonal signaling.

ABSTRACT The histone H3 lysine 79 methyltransferase DOT1L/KMT4 can promote an oncogenic pattern of gene expression through binding with several MLL fusion partners found in acute leukemia. However, the normal function of DOT1L in mammalian gene regulation is poorly understood. Here we report that DOT1L recruitment is ubiquitously coupled with active transcription in diverse mammalian cell types. DOT1L preferentially occupies the proximal transcribed region of active genes, correlating with enrichment of H3K79 di- and trimethylation. Furthermore, Dot1l mutant fibroblasts lacked H3K79 di- and trimethylation at all sites examined, indicating that DOT1L is the sole enzyme responsible for these marks. Importantly, we identified chromatin immunoprecipitation (ChIP) assay conditions necessary for reliable H3K79 methylation detection. ChIP-chip tiling arrays revealed that levels of all degrees of genic H3K79 methylation correlate with mRNA abundance and dynamically respond to changes in gene activity. Conversion of H3K79 monomethylation into di- and trimethylation correlated with the transition from low- to high-level gene transcription. We also observed enrichment of H3K79 monomethylation at intergenic regions occupied by DNA-binding transcriptional activators. Our findings highlight several similarities between the patterning of H3K4 methylation and that of H3K79 methylation in mammalian chromatin, suggesting a widespread mechanism for parallel or sequential recruitment of DOT1L and MLL to genes in their normal “on” state.

Abstract Abstract 2467 NUP98-fusions although rare, have been associated with de novo acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, T-cell acute lymphoblastic leukemia (T-ALL) and therapy-related myeloid malignancies. The NUP98-PHF23 (NP23) gene fusion was cloned from an acute myeloid leukemia (AML) patient with a t(11;17)(p15;p13) chromosome translocation. The nucleoporin 98 protein (NUP98), normally a component of the nuclear pore complex, is known to be fused to at least 28 different fusion partners as a result of structural chromosomal rearrangements associated with hematological malignancies. PHF23 encodes the Plant homeodomain (PHD) finger 23 protein. PHF23 is largely uncharacterized, but the PHD finger motif has been shown to act as a reader of di- and tri-methylated histone 3 lysine 4 (H3K4me2/3) marks. This suggests that PHF23 may function in chromatin regulation and that the NP23 fusion protein may play a role in aberrant chromatin modification at domains of active gene transcription. To determine the oncogenic potential of NP23, we generated a transgenic mouse model that expressed the human fusion gene in hematopoietic tissues. We have characterized two founder lines (C10 and B10) expressing the NP23 fusion in hematopoietic tissue. Most of the offspring from the C10 line developed an AML that closely resembled the human disease, with increased blasts in the blood or bone marrow, widespread organ infiltration, and myeloid immunophenotype. Onset of disease was as early as 4.5 months, and 70 percent of the NP23 mice succumbed to leukemia by 12 months of age. Of note, an independent line (B10) developed a wider spectrum of leukemias but with similar age of onset and penetrance. The B10 mice predominantly developed T-ALL and AML, and four cases of B-ALL and one erythroleukemia, indicating that the NP23 protein was oncogenic in several different hematopoietic cell types. AMLs typically demonstrated an aberrant Mac-1+/B220dim phenotype, which has previously been recognized in leukemias caused by overexpression of the Hoxa cluster genes Hoxa5,7,9,10,11. Microarray gene expression analysis identified the Hoxa cluster genes to be markedly overexpressed, and validation by RQ-PCR demonstrated that Hoxa5, a7, a9 and a10 overexpression ranged from 10- to greater than 1000-fold increased in both the AML and T-ALL samples compared to wild type hematopoietic tissues; these Hoxa cluster genes were also overexpressed in hematopoietic tissues from clinically healthy NP23 transgenic mice. We also identified a novel transcript, Gm525 (homologue of H. sapiens C17orf67), that is markedly (100x) elevated specifically in the T-ALL samples. Most T-ALL samples have HD or PEST domain Notch1 mutations, and Notch1 mRNA levels, as well as its downstream target Hes1, are elevated in the T-ALLs compared to WT thymus. Immortal cell lines were established from two of the NP23 T-ALLs, and ChIP-seq was used to assay the genome wide pattern of H3K4me3 and H3K27me3 histone marks. Results show abundant levels of H3K4me3 at the Hoxa locus which tightly correlates to the increased Hoxa cluster gene expression seen in the cell lines. The NP23 model will be useful for identifying oncoproteins involved in leukemic transformation, particularly those oncoproteins which play a role in chromatin modification or are downstream targets of the HOXA genes. Disclosures: No relevant conflicts of interest to declare.

Bacterial biofilms are large aggregates of cells embedded in an extracellular matrix of self-produced polymers. In macrocolony biofilms of Escherichia coli , this matrix is generated in the upper biofilm layer only and shows a surprisingly complex supracellular architecture. Stratified matrix production follows the vertical nutrient gradient and requires the stationary phase σ S (RpoS) subunit of RNA polymerase and the second messenger c-di-GMP. By visualizing global gene expression patterns with a newly designed fingerprint set of Gfp reporter fusions, our study reveals the spatial order of differential sigma factor activities, stringent control of ribosomal gene expression and c-di-GMP signalling in vertically cryosectioned macrocolony biofilms. Long-range physiological stratification shows a duplication of the growth-to-stationary phase pattern that integrates nutrient and oxygen gradients. In addition, distinct short-range heterogeneity occurs within specific biofilm strata and correlates with visually different zones of the refined matrix architecture. These results introduce a new conceptual framework for the control of biofilm formation and demonstrate that the intriguing extracellular matrix architecture, which determines the emergent physiological and biomechanical properties of biofilms, results from the spatial interplay of global gene regulation and microenvironmental conditions. Overall, mature bacterial macrocolony biofilms thus resemble the highly organized tissues of multicellular organisms.

ABSTRACT Alginate biosynthesis by Pseudomonas aeruginosa was shown to be regulated by the intracellular second messenger bis-(3′-5′)-cyclic-dimeric-GMP (c-di-GMP), and binding of c-di-GMP to the membrane protein Alg44 was required for alginate production. In this study, PA1727, a c-di-GMP-synthesizing enzyme was functionally analyzed and identified to be involved in regulation of alginate production. Deletion of the PA1727 gene in the mucoid alginate-overproducing P. aeruginosa strain PDO300 resulted in a nonmucoid phenotype and an about 38-fold decrease in alginate production; thus, this gene is designated mucR. The mucoid alginate-overproducing phenotype was restored by introducing the mucR gene into the isogenic ΔmucR mutant. Moreover, transfer of the MucR-encoding plasmid into strain PDO300 led to an about sevenfold increase in alginate production, wrinkly colony morphology, increased pellicle formation, auto-aggregation, and the formation of highly structured biofilms as well as the inhibition of swarming motility. Outer membrane protein profile analysis showed that overproduction of MucR mediates a strong reduction in the copy number of FliC (flagellin), required for flagellum-mediated motility. Translational reporter enzyme fusions with LacZ and PhoA suggested that MucR is located in the cytoplasmic membrane with a cytosolic C terminus. Deletion of the proposed C-terminal GGDEF domain abolished MucR function. MucR was purified and identified using tryptic peptide fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Overall, experimental evidence was provided suggesting that MucR specifically regulates alginate biosynthesis by activation of alginate production through generation of a localized c-di-GMP pool in the vicinity of Alg44.

Mixed Lineage Leukemia 1 (MLL1), an important member of Histone Methyltransferases (HMT) family, is capable of catalyzing mono-, di-, and trimethylation of Histone 3 lysine 4 (H3K4). The optimal catalytic activity of MLL1 requires the formation of a core complex consisting of MLL1, WDR5, RbBP5, and ASH2L. The Protein-Protein Interaction (PPI) between WDR5 and MLL1 plays an important role in abnormal gene expression during tumorigenesis, and disturbing this interaction may have a potential for the treatment of leukemia harboring MLL1 fusion proteins. In this review, we will summarize recent progress in the development of inhibitors targeting MLL1- WDR5 interaction.

Abstract The clustered Hoxa genes Hoxa5-Hoxa10 are important for self-renewal and differentiation of hematopoietic stem and progenitor cells. These genes are often aberrant upregulated in human malignancies, one of the most striking examples of which are leukemias with rearrangements of the mixed lineage leukemia (MLL) gene. Since these key posterior Hoxa cluster genes are known to show graded down-regulation upon hematopoietic differentiation, we sought to assess the epigenetic changes associated with this tightly controlled, developmentally critical transcriptional program. We performed ChIP sequencing on lineage negative, Sca-1 + Kit + (LSK) cells where the posterior Hoxa5-10genes are highly expressed compared to granulocyte macrophage progenitors (GMPs) that show markedly lower expression of these transcripts. We observed a dramatic diminution in H3K79 di/tri methylation as cells differentiated from LSKs to GMPs. In contrast, we found minimal changes in H3K79 mono-methylation during the LSK-GMP transition suggesting that high expression of Hoxa genes is maintained through higher H3K79 methylated states in normal hematopoiesis. Strikingly, the removal of H3K79 me2/3 but not me1 was sufficient for repressive epigenetic mechanisms such as polycomb repressive complex 2 (PRC2)- mediated H3K27 tri-methylation to invade the Hoxa locus during the LSK to GMP differentiation. The decrease in H3K79 di/tri methylation upon hematopoietic differentiation may be the result of a graded decrease in expression of the DOT1L co-factor Af10 which we have recently found to regulate the transition from H3K79 mono to dimethylation. Next we wanted to probe whether MLL-leukemias drive Hoxa gene expression through increased conversion of H3K79 methylation from the mono to the di/tri-methylated states. We performed ChIP sequencing for H3K79 me1/2/3 to identify the different states of H3K79 methylation across the epigenetic landscape of MLL-transformed cells. Strikingly we observed that in both murine and human leukemia, there was a dramatic hyper-conversion of H3K79 mono-methylation to di/trimethylation specifically at MLL-AF9 target genes. Af10 deletion in the MLL-AF9 leukemia significantly reduced H3K79me2/3 while retaining - and in some cases even increasing - H3K79 me1 at MLL-target genes. We then assessed chromatin accessibility at the Hoxa locus following nuclease digestion and observed that the diminution of H3K79me2/3 was enough to significantly reduce chromatin accessibility at the Hoxa locus despite the relative retention of H3K79me1. Similar to the LSK-GMP transition, reduction in H3K79 me2/3 but not H3K79me1 was accompanied by a dramatic increase in H3K27me3 in a very specific subset of genes including Hoxa5-10. The genetic and epigenetic changes resulting from Af10 deletion significantly impaired MLL-leukemogenesis and sensitized leukemia cells to DOT1L inhibition. These results suggest that the expression of Hoxa genes in hematopoiesis is controlled through higher H3K79 methylated states, possibly aided by the DOT1L co-factor Af10. The MLL-AF9 fusion seems to hijack this mode of regulation through aberrant hyperconversion of H3K79 methylation to higher methylated states at critical oncogenic targets including the Hoxa genes. These data suggest that targeting the interaction between DOT1L and Af10 is a potential therapeutic strategy since it blunts the conversion of H3K79me1 to higher states of methylation by MLL-fusion proteins and thus significantly impairs MLL-mediated transformation. Disclosures: Armstrong: Epizyme: Consultancy.

Abstract Epigenetic abnormalities are frequently involved in the initiation and progression of cancers, including acute myeloid leukemia (AML). A subtype of AML, acute promyelocytic leukemia (APL), is mainly driven by a specific oncogenic fusion event of promyelocytic leukemia–RA receptor fusion oncoprotein (PML-RARα). PML-RARα was reported as a transcription repressor through the interaction with nuclear receptor corepressor and histone deacetylase complexes leading to the mis-suppression of its target genes and differentiation blockage. Although previous studies were mainly focused on the connection of histone acetylation, it is still largely unknown whether alternative epigenetics mechanisms are involved in APL progression. KDM5A is a demethylase of histone H3 lysine 4 di- and tri-methylations (H3K4me2/3) and a transcription corepressor. Here, we found that the loss of KDM5A led to APL NB4 cell differentiation and retarded growth. Mechanistically, through epigenomics and transcriptomics analyses, KDM5A binding was detected in 1889 genes, with the majority of the binding events at promoter regions. KDM5A suppressed the expression of 621 genes, including 42 PML-RARα target genes, primarily by controlling the H3K4me2 in the promoters and 5′ end intragenic regions. In addition, a recently reported pan-KDM5 inhibitor, CPI-455, on its own could phenocopy the differentiation effects as KDM5A loss in NB4 cells. CPI-455 treatment or KDM5A knockout could greatly sensitize NB4 cells to all-trans retinoic acid–induced differentiation. Our findings indicate that KDM5A contributed to the differentiation blockage in the APL cell line NB4, and inhibition of KDM5A could greatly potentiate NB4 differentiation.

Abstract The mixed lineage leukemia (MLL) protein is a histone methyltransferase that writes the histone H3 lysine 4 trimethyl (H3K4me3) mark at the promoters of target genes such as HOXA9 and MEIS1. MLL is the target of chromosomal translocations that fuse it in frame to one of over 90 partners, leading to acute myeloid and lymphoid leukemias (AML and ALL, respectively) characterized by poor prognoses1. MLL fusions activate transcription by recruiting the AF4 family/ENL family/P-TEFb (AEP) complex and the DOT1L-AF10 family-ENL family complex (DOT1L complex or DotCom). Transcriptional activation via AF4 recruitment and transcriptional maintenance via DOT1L recruitment are required for MLL leukemias. Despite the large number of fusion partners, members of the AEP complex account for nearly 70% of MLL rearrangements1. These fusions constitutively activate MLL targets by bypassing recruitment via ENL (MLLT1) and AF9 (MLLT3) YEATS domain binding to crotonylated or acetylated histone H3. The AF9 ANC1 homology domain (AHD), retained in MLL fusions, is intrinsically disordered, but undergoes coupled folding and binding upon interaction with its binding proteins2. The AHD recruits AF4 and DOT1L, which support transcriptional elongation, as well as the BCL6 corepressor (BCOR) and chromobox homolog 8 (CBX8), which are implicated in transcriptional repression. CBX8 (HPC3) is a mammalian ortholog of Drosophila polycomb that binds trimethylated histone H3 lysine 9 and 27 (H3K9me3 and H3K27me3) with variable affinity. Previous reports indicate CBX8 is required for MLL-AF9 and MLL-ENL. BCOR is a transcriptional corepressor that augments BCL6-mediated repression. The BCL6 POZ domain forms a ternary complex with BCOR and SMRT, repressing targets via recruitment of PRC1.1 and HDAC3. BCOR translocations and mutations have been found in a range of cancers. Although it is broadly expressed throughout the hematopoietic system (Bloodspot), little is known about BCOR function in hematopoiesis. Recently, BCOR was shown to have a role in maintenance of human embryonic stem cell pluripotency. BCOR has also been implicated in regulation of myeloid cell proliferation and differentiation and is necessary for MLL-AF9 leukemogenesis. While the roles of the direct MLL-AF9/AF4 and MLL-AF9/DOT1L interactions have been the subject of previous structural and functional studies2-4, the roles of the direct interactions of MLL-AF9 with CBX8 and BCOR remain relatively uncharacterized. We determined the structures of the AF9 AHD-CBX8 and AF9 AHD-BCOR complexes. Based on the structures, we developed point mutants to increase and decrease affinity of CBX8 for AF9. Increased affinity decreased colony forming ability and induced differentiation of MLL-AF9-transformed cells, while decreased affinity had no effect. An additional point mutant was developed to selectively disrupt BCOR binding to AF9. In the context of MLL-AF9, this mutant increases proliferative ability without an effect on colony formation and is unable to cause leukemia in vivo. RNAseq analysis reveals that this mutant affects a different set of genes than loss of DOT1L or AF4 binding or gain of CBX8 binding, leading to a phenotype distinct from that seen with perturbation of other AF9 interactions, functionally distinguishing proliferative capacity from in vivo leukemogenesis. In particular, substantial effects were observed on EYA1 expression, suggesting a critical role for the EYA1/SIX gene network in MLL-AF9 leukemia. 1 Meyer, C. et al. The MLL recombinome of acute leukemias in 2017. Leukemia32, 273-284, doi:10.1038/leu.2017.213 (2018). 2 Leach, B. I. et al. Leukemia fusion target AF9 is an intrinsically disordered transcriptional regulator that recruits multiple partners via coupled folding and binding. Structure21, 176-183, doi:10.1016/j.str.2012.11.011 (2013). 3 Kuntimaddi, A. et al. Degree of recruitment of DOT1L to MLL-AF9 defines level of H3K79 Di- and tri-methylation on target genes and transformation potential. Cell reports11, 808-820, doi:10.1016/j.celrep.2015.04.004 (2015). 4 Lokken, A. A. et al. Importance of a specific amino acid pairing for murine MLL leukemias driven by MLLT1/3 or AFF1/4. Leukemia research38, 1309-1315, doi:10.1016/j.leukres.2014.08.010 (2014). Disclosures No relevant conflicts of interest to declare.

Lo studio si è basato su tecnologia di SNP array per identificare regioni cromosomiche oggetto di alterazioni di copy number in un’ampia casisitica di pazienti con neoplasie mieloproliferative croniche Ph-negative, sia nella fase cronica di malattia sia soprattutto nella transizione ad una leucemia acuta. Sulla base di una di queste anomalie identificate, sono poi state condotte ulteriori ricerche che hanno permesso di identificare una nuova anomalia molecolare, non descritta in precedenza, che genera un trascritto chimerico di fusione che è in avanzata fase di caratterizzazione. In this study SNP array technology was used to identify chromosomal regions subject to copy number alterations in a wide number of patients with Ph-negative chronic myeloproliferative neoplasms, both in the chronic phase of the disease and in the transition to acute leukemia. On the basis of these first results, further studies were conducted that allow to identified a new molecular defect, not previously described, which generates a chimeric fusion transcript that is in an advanced stage of characterization.