Articles de revues : « Gene di fusione »

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Padella, Antonella, Giorgia Simonetti, Giulia Paciello, Anna Ferrari, Elisa Zago, Carmen Baldazzi, Viviana Guadagnuolo et al. « RNA Sequencing Reveals Novel and Rare Fusion Transcripts in Acute Myeloid Leukemia ». Blood 126, n^o 23 (3 décembre 2015) : 3627. http://dx.doi.org/10.1182/blood.v126.23.3627.3627.

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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.

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Song, Xiaowen, Qisheng Zhong, Guifang Peng, Yanhao Ji, Yuemei Zhang, Jing Tang, Jia Xie, Jingxiu Bi, Fan Feng et Bin Li. « Functional characterization of a special dicistronic transcription unit encoding histone methyltransferase su(var)3-9 and translation regulator eIF2γ in Tribolium castaneum ». Biochemical Journal 477, n^o 16 (28 août 2020) : 3059–74. http://dx.doi.org/10.1042/bcj20200444.

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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.

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Steger, David J., Martina I. Lefterova, Lei Ying, Aaron J. Stonestrom, Michael Schupp, David Zhuo, Adam L. Vakoc et al. « DOT1L/KMT4 Recruitment and H3K79 Methylation Are Ubiquitously Coupled with Gene Transcription in Mammalian Cells ». Molecular and Cellular Biology 28, n^o 8 (19 février 2008) : 2825–39. http://dx.doi.org/10.1128/mcb.02076-07.

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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.

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Gough, Sheryl M., Fan Lee, Yang Jo Chung, Robert L. Walker, Fan Yang, Yuelin (Jack) Zhu, Yi Ning, Paul S. Meltzer et Peter Aplan. « A NUP98-PHF23 Transgenic Mouse Model Develops AML and T-ALL ». Blood 118, n^o 21 (18 novembre 2011) : 2467. http://dx.doi.org/10.1182/blood.v118.21.2467.2467.

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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.

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Klauck, Gisela, Diego O. Serra, Alexandra Possling et Regine Hengge. « Spatial organization of different sigma factor activities and c-di-GMP signalling within the three-dimensional landscape of a bacterial biofilm ». Open Biology 8, n^o 8 (août 2018) : 180066. http://dx.doi.org/10.1098/rsob.180066.

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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.

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Hay, Iain D., Uwe Remminghorst et Bernd H. A. Rehm. « MucR, a Novel Membrane-Associated Regulator of Alginate Biosynthesis in Pseudomonas aeruginosa ». Applied and Environmental Microbiology 75, n^o 4 (16 décembre 2008) : 1110–20. http://dx.doi.org/10.1128/aem.02416-08.

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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.

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Ye, Xiaoqing, Gang Chen, Jia Jin, Binzhong Zhang, Yinda Wang, Zhenhai Cai et Fei Ye. « The Development of Inhibitors Targeting the Mixed Lineage Leukemia 1 (MLL1)-WD Repeat Domain 5 Protein (WDR5) Protein- Protein Interaction ». Current Medicinal Chemistry 27, n^o 33 (8 octobre 2020) : 5530–42. http://dx.doi.org/10.2174/0929867326666190528080514.

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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.

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Deshpande, Aniruddha J., Liying Chen, Amit U. Sinha, Nan Zhu, David Chen, Jenny Chang, Andrei V. Krivtsov, Kathrin Bernt, James E. Bradner et Scott A. Armstrong. « Regulation Of Normal and Malignant Hoxa Gene Expression Through Higher H3K79 Methylated States ». Blood 122, n^o 21 (15 novembre 2013) : 2492. http://dx.doi.org/10.1182/blood.v122.21.2492.2492.

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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.

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Xu, Siyuan, Siqing Wang, Shenghui Xing, Dingdang Yu, Bowen Rong, Hai Gao, Mengyao Sheng et al. « KDM5A suppresses PML-RARα target gene expression and APL differentiation through repressing H3K4me2 ». Blood Advances 5, n^o 17 (27 août 2021) : 3241–53. http://dx.doi.org/10.1182/bloodadvances.2020002819.

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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.

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Bushweller, John H., Charles Schmidt, Nicholas Achille, Aravinda Kuntimaddi, Adam Boulton, Benjamin Leach, Shubin Zhang et Nancy J. Zeleznik-Le. « Direct Binding of BCOR, but Not CBX8, to MLL-AF9 Is Essential for MLL-AF9 Leukemia Via Regulation of the EYA1/SIX1 Gene Network ». Blood 132, Supplement 1 (29 novembre 2018) : 1316. http://dx.doi.org/10.1182/blood-2018-99-111388.

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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.

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Boer, Judith M., Maria Grazia Valsecchi, Femke M. Hormann, Zeljko Antic, Marketa Zaliova, Claire Schwab, Giovanni Cazzaniga et al. « NUTM1-Rearranged Infant and Pediatric B Cell Precursor Acute Lymphoblastic Leukemia : A Good Prognostic Subtype Identified in a Collaborative International Study ». Blood 136, Supplement 1 (5 novembre 2020) : 25–26. http://dx.doi.org/10.1182/blood-2020-139376.

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Background and Aims A novel genetic subtype of B cell precursor acute lymphoblastic leukemia (B-ALL) is characterized by rearrangement of NUTM1 (NUTM1r) on 15q14 resulting in fusion of NUTM1 to one of several partner genes such as CUX1, ACIN1, BRD9, and IKZF1. The downstream effects of NUTM1r include upregulation of the proto-oncogene BMI1 and specific fusions also induce transcription of the HOXA gene cluster (Hormann et al. Haematologica 2019; Li et al. PNAS 2018). This novel subtype is rare in children, but appears to be more prevalent among infants negative for KMT2A rearrangement (KMT2Ar) based on the frequency of karyotypic 15q aberrations (De Lorenzo et al. Leukemia 2014). This international collaborative study aimed to determine the frequency of NUTM1r in infant and pediatric cohorts, and to characterize the demographic, clinical and molecular features of NUTM1r-positive B-ALL. Patients and Methods Interfant-related study groups provided NUTM1 screening results for KMT2Ar-negative Interfant-99 and -06 cases with karyotypic 15q aberration, normal karyotype, or missing karyotype. Additionally, NUTM1r-positive cases of any age were collected from the study groups united in the Ponte di Legno consortium. The identified NUTM1r-positive children were diagnosed between 1995-2019, infants (≤365 days of age) included in the Interfant-99 or 06 trials were diagnosed between 2000-2016, and remaining infants between 1986-2019. The techniques used for the detection of NUTM1r were break-apart FISH, RNA sequencing, and RT-PCR. Event-free survival (EFS) and overall survival (OS) were estimated according to Kaplan-Meier, standard error according to Greenwood, and the curves were compared by log-rank test. Results We identified 81 NUTM1r cases, including 35 Interfant-enrolled infants, 10 other infants and 36 children. NUTM1r was reported to be rare among pediatric B-ALL with an estimated frequency range of 0.28-0.86%. The median age among NUTM1r-positive children was 4.5 years (range 1-15). Among KMT2Ar-negative infants the frequency of NUTM1r was 21.7%. Of NUTM1r-positive infants, 54% were <6 months at diagnosis (median 5.6, range 0.4-11.0 months) compared with 16% in the remaining KMT2Ar-negative infants (median 9.3, range 0.1-11.9; p<0.0001). Other baseline characteristics (WBC, gender, prednisone poor response) were similar between NUTM1r-positive and -negative infants. Of the NUTM1r-positive cases, all achieved complete remission, 82% had minimal residual disease <10e-4 at the end of induction, and no patient received stem cell transplant in first remission. The 4-year EFS was 100% in Interfant-enrolled NUTM1r-positive patients versus 74% (95% CI 65.1-81.0, p=0.001) in the remaining KMT2Ar-negative cases (n=126). The better outcome was confirmed also after adjusting for WBC, gender and prednisone response (p=0.0001). The 4-year OS were 100% and 88.0% (95% CI 80.5-92.7) for NUTM1r-positive and other KMT2Ar-negative infant cases, respectively (p-value=0.04). Children and non-Interfant-enrolled infants treated on different treatment protocols showed 89.4% (95% CI 78.6-1) 4-year EFS and 100% 4-year OS. In order of frequency, NUTM1 fusion partners were ACIN1 (30.4%), CUX1 (21.7%), BRD9 (17.4%), ZNF618 (13%), AFF1 (4.3%), SLC12A6 (4.3%), IKZF1 (2.9%), and three novel partners: ATAD5 (2.9%), CHD4 (1.4%) and RUNX1 (1.4%). Infants mainly showed fusions with ACIN1, CUX1, BRD9 and AFF1, associated with HOXA9 upregulation. Older infants and children showed both HOXA-upregulating and non-HOXA-upregulating fusions. Epigenetic profiling showed a distinct pattern of DNA methylation and histone modification of the HOXA gene cluster region in leukemic cells of an ACIN1-NUTM1 pediatric case compared with KMT2Ar-positive and KMT2Ar/NUTM1r-negative pediatric cases. Conclusions NUTM1r ALL was identified as the second largest subtype in infants, found in 21.7% of KMT2Ar-negative infant B-ALL, representing 5-7% of total infant ALL, and associated with excellent outcome on Interfant standard risk protocols. The favorable outcome was confirmed in the Ponte di Legno cohort of infant and pediatric NUTM1r-positive patients enrolled on different treatment protocols over more than two decades. We conclude that NUTM1r ALL is a favorable genetic subtype in infants and children and possibly eligible for treatment reduction. Disclosures No relevant conflicts of interest to declare.

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Ibrahim, Eid I., Kotb A. Attia, Abdelhalim I. Ghazy, Kimiko Itoh, Fahad N. Almajhdi et Abdullah A. Al-Doss. « Molecular Characterization and Functional Localization of a Novel SUMOylation Gene in Oryza sativa ». Biology 11, n^o 1 (31 décembre 2021) : 53. http://dx.doi.org/10.3390/biology11010053.

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Small ubiquitin-related modifier (SUMO) regulates the cellular function of diverse proteins through post-translational modifications. The current study defined a new homolog of SUMO genes in the rice genome and named it OsSUMO7. Putative protein analysis of OsSUMO7 detected SUMOylation features, including di-glycine (GG) and consensus motifs (ΨKXE/D) for the SUMOylation site. Phylogenetic analysis demonstrated the high homology of OsSUMO7 with identified rice SUMO genes, which indicates that the OsSUMO7 gene is an evolutionarily conserved SUMO member. RT-PCR analysis revealed that OsSUMO7 was constitutively expressed in all plant organs. Bioinformatic analysis defined the physicochemical properties and structural model prediction of OsSUMO7 proteins. A red fluorescent protein (DsRed), fused with the OsSUMO7 protein, was expressed and localized mainly in the nucleus and formed nuclear subdomain structures. The fusion proteins of SUMO-conjugating enzymes with the OsSUMO7 protein were co-expressed and co-localized in the nucleus and formed nuclear subdomains. This indicated that the OsSUMO7 precursor is processed, activated, and transported to the nucleus through the SUMOylation system of the plant cell.

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Di Gregorio, A., et M. Levine. « Regulation of Ci-tropomyosin-like, a Brachyury target gene in the ascidian, Ciona intestinalis ». Development 126, n^o 24 (15 décembre 1999) : 5599–609. http://dx.doi.org/10.1242/dev.126.24.5599.

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Brachyury is a sequence-specific transcriptional activator that is essential for notochord differentiation in a variety of chordates. In vertebrates, Brachyury is expressed throughout the presumptive mesoderm, but becomes restricted to the notochord at later stages of development. In ascidians, such as Ciona intestinalis, Brachyury is expressed exclusively in the notochord and does not exhibit an early pan-mesodermal pattern. Subtractive hybridization screens were recently used to identify potential Ciona Brachyury (Ci-Bra) target genes (Takahashi, H., Hotta, K., Erives, A., Di Gregorio, A., Zeller, R. W., Levine, M. and Satoh, N. (1999). Genes Dev. 13, 1519–1523). Of the genes that were identified in this screen, one corresponds to a new member of the tropomyosin superfamily, Ciona tropomyosin (Ci-trop). Here we show that Ci-trop is specifically expressed in the developing notochord beginning at gastrulation, and expression persists in the notochord during tailbud and tadpole stages. A 3 kb region of the Ci-trop 5′-flanking sequence was characterized via electroporation of lacZ fusion genes into fertilized Ciona eggs. A minimal, 114 bp enhancer was identified that is sufficient to direct the expression of a heterologous promoter in the notochord. DNA binding assays indicate that this enhancer contains two sets of low-affinity Brachyury half-sites, which are bound in vitro by a GST/Ci-Bra fusion protein. Deletion of the distal sites inactivates the notochord-specific staining pattern mediated by an otherwise normal Ci-trop/lacZ transgene. These results suggest that Ci-trop is a direct target gene of Ci-Bra and that Brachyury plays an immediate role in the cellular morphogenesis of the notochord.

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Boustead, JN, CC Martin, JK Oeser, CA Svitek, SI Hunter, JC Hutton et RM O'Brien. « Identification and characterization of a cDNA and the gene encoding the mouse ubiquitously expressed glucose-6-phosphatase catalytic subunit-related protein ». Journal of Molecular Endocrinology 32, n^o 1 (1 février 2004) : 33–53. http://dx.doi.org/10.1677/jme.0.0320033.

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Glucose-6-phosphatase (G6Pase) catalyzes the final step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate. This paper describes the identification and characterization of a cDNA and the gene encoding the mouse ubiquitously expressed G6Pase catalytic subunit-related protein (UGRP). The open reading frame of this UGRP cDNA encodes a protein (346 amino acids (aa); Mr 38,755) that shares 36% overall identity (56% similarity) with the mouse G6Pase catalytic subunit (357 aa; Mr 40,454). UGRP exhibits a similar predicted transmembrane topology and conservation of many of the catalytically important residues with the G6Pase catalytic subunit; however, unlike the G6Pase catalytic subunit, UGRP does not catalyze G6P hydrolysis and does not contain a carboxy-terminal di-lysine endoplasmic reticulum retention signal. UGRP mRNA was detected by RNA blot analysis in every mouse tissue examined with the highest expression in heart, brain, testis and kidney. Database analysis showed that the mouse UGRP gene is composed of six exons, spans approximately 4.2 kbp of genomic DNA and is located on chromosome 11 along with the G6Pase catalytic subunit gene. The UGRP gene transcription start sites were mapped by primer extension analysis, and the activity of the mouse UGRP gene promoter was analyzed using luciferase fusion gene constructs. In contrast to the G6Pase catalytic subunit gene promoter, the UGRP promoter was highly active in all cell lines examined.

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Wozniak, Christopher E., Changhan Lee et Kelly T. Hughes. « T-POP Array Identifies EcnR and PefI-SrgD as Novel Regulators of Flagellar Gene Expression ». Journal of Bacteriology 191, n^o 5 (29 décembre 2008) : 1498–508. http://dx.doi.org/10.1128/jb.01177-08.

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ABSTRACT The T-POP transposon was employed in a general screen for tetracycline (Tet)-induced chromosomal loci that exhibited Tet-activated or Tet-repressed expression of a fliC-lac transcriptional fusion. Insertions that activated flagellar transcription were located in flagellar genes. T-POP insertions that exhibited Tet-dependent fliC-lac inhibition were isolated upstream of the ecnR, fimZ, pefI-srgD, rcsB, and ydiV genes and in the flagellar gene flgA, which is located upstream of the anti-σ28 factor gene flgM. When expressed from the chromosomal P araBAD promoter, EcnR, FimZ, PefI-SrgD, and RcsB inhibited the transcription of the flagellar class 1 flhDC operon. YdiV, which is weakly homologous to EAL domain proteins involved in cyclic-di-GMP regulation, appears to act at a step after class 1 transcription. By using a series of deletions of the regulatory genes to try to disrupt each pathway, these regulators were found to act largely independently of one another. These results identify EcnR and PefI-SrgD as additional components of the complex regulatory network controlling flagellar expression.

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Apfel, Christian M., Béla Takács, Michael Fountoulakis, Martin Stieger et Wolfgang Keck. « Use of Genomics To Identify Bacterial Undecaprenyl Pyrophosphate Synthetase : Cloning, Expression, and Characterization of the Essential uppS Gene ». Journal of Bacteriology 181, n^o 2 (15 janvier 1999) : 483–92. http://dx.doi.org/10.1128/jb.181.2.483-492.1999.

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ABSTRACT The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC2.5.1.31 ) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E,E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphos-phate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coliproteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675 ; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and inCaenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli,Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.

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Wiethaus, Jessica, Alexandra Müller, Meina Neumann, Sandra Neumann, Silke Leimkühler, Franz Narberhaus et Bernd Masepohl. « Specific Interactions between Four Molybdenum-Binding Proteins Contribute to Mo-Dependent Gene Regulation in Rhodobacter capsulatus ». Journal of Bacteriology 191, n^o 16 (5 juin 2009) : 5205–15. http://dx.doi.org/10.1128/jb.00526-09.

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ABSTRACT The phototrophic purple bacterium Rhodobacter capsulatus encodes two transcriptional regulators, MopA and MopB, with partially overlapping and specific functions in molybdate-dependent gene regulation. Both MopA and MopB consist of an N-terminal DNA-binding helix-turn-helix domain and a C-terminal molybdate-binding di-MOP domain. They formed homodimers as apo-proteins and in the molybdate-bound state as shown by yeast two-hybrid (Y2H) studies, glutaraldehyde cross-linking, gel filtration chromatography, and copurification experiments. Y2H studies suggested that both the DNA-binding and the molybdate-binding domains contribute to dimer formation. Analysis of molybdate binding to MopA and MopB revealed a binding stoichiometry of four molybdate oxyanions per homodimer. Specific interaction partners of MopA and MopB were the molybdate transporter ATPase ModC and the molbindin-like Mop protein, respectively. Like other molbindins, the R. capsulatus Mop protein formed hexamers, which were stabilized by binding of six molybdate oxyanions per hexamer. Heteromer formation of MopA and MopB was shown by Y2H studies and copurification experiments. Reporter gene activity of a strictly MopA-dependent mop-lacZ fusion in mutant strains defective for either mopA, mopB, or both suggested that MopB negatively modulates expression of the mop promoter. We propose that depletion of the active MopA homodimer pool by formation of MopA-MopB heteromers might represent a fine-tuning mechanism controlling mop gene expression.

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Hodges, Vivien M., Susan Rainey, Victoria Smyth, Ken Mills, Terence R. J. Lappin et A. Peter Maxwell. « PHF23 : A Novel Erythropoietin-Induced Gene Associated with AML and MDS. » Blood 110, n^o 11 (16 novembre 2007) : 3178. http://dx.doi.org/10.1182/blood.v110.11.3178.3178.

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Abstract Chromosomal translocations in a number of hematopoietic malignancies, including AML and MDS, result in the formation of NUP98 gene chimeras. Recently, fusion of NUP98 with a novel gene, PHF23 or plant homeodomain (PHD) finger 23, was reported in a cryptic translocation in a patient with AML (Reader et al., 2007). PHF23 is one of three known NUP98 fusion partners, associated with AML, which contain PHD finger motifs. In this study, we report a more detailed characterization of PHF23, which we originally identified by differential display PCR as an erythropoietin-induced gene in a murine model of terminal erythroid differentiation. In silico sequence analysis revealed a five exon gene containing a nuclear localization signal (NLS) as well as the C-terminal PHD motif. PHF23 mapped to chromosome 17p13.1, <450 kb from the TP53 gene, in a region frequently disrupted in cancer. Quantitative PCR analysis indicated Phf23 expression is not restricted to hematopoietic cells but demonstrates widespread tissue distribution, with increased expression levels in more proliferative tissues (∼6 fold increase in gene expression in the testis over mRNA levels in the liver). A ubiquitously expressed Phf23 splice variant (with mRNA concentrations ∼30–45% lower than full-length Phf23 gene expression levels) was generated using an alternative 3′ splice site within exon four, which exactly corresponded to the chr17 translocation breakpoint in the AML patient described. PHF23 is evolutionarily conserved, particularly within the PHD finger domain (>76% sequence identity between human and zebrafish PHD motifs), and is significantly related to another potentially oncogenic protein, PHF13/SPOC-1, associated with ovarian cancer. PHD motifs act as zinc fingers and the expanding family of PHD-containing proteins are implicated in chromatin regulation. Cellular localization studies using fluorescently tagged PHF23 indicated that both the full-length and spliced isoforms are located exclusively within the nucleus. We predicted that PHF23 has a similar structure to PHD motifs of known histone effector proteins, which influence transcription through recognition of specific epigenetic marks. This was demonstrated by immunoprecipitation and histone peptide binding assays showing that PHF23 associated with histone H3, primarily di- and tri-methylated H3K4, which are characteristic marks of active transcription. Microarray analysis of PHF23 mRNA expression levels in AML and MDS patient samples indicated no defined association with AML, however a positive correlation was demonstrated with a subset of MDS patients exhibiting erythroid dysplasia. This study suggests that PHF23, a novel histone effector protein, may play a role in aberrant transcription during defective hematopoiesis.

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Staak, Jan O., David Colcher, Scott Lauder, Jianyi Wang, Stephen D. Gillies et Andrew A. Raubitschek. « The Novel De-Immunized Anti-CD20-IL-2 Immunocytokine DI-Leu16-IL-2 Is Effective in SCID Mice Bearing Rituximab-Resistant Human Lymphoma Xenografts. » Blood 104, n^o 11 (16 novembre 2004) : 3288. http://dx.doi.org/10.1182/blood.v104.11.3288.3288.

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Abstract Objectives: Tumor-targeted MAb-cytokine fusion proteins, such as IL-2 based immunocytokines, have demonstrated potent anti-tumor activity in several pre-clinical models as well as early stage clinical trials. For the engineering of anti-CD20-IL-2, the parental murine anti-CD20 MAb Leu16 was deimmunized by removal of potential helper T cell epitopes and then humanized by combination of recombinant V regions with human L chain and H chain constant regions. Fusion of the H chain to human IL-2 resulted in the de-immunized immunocytokine DI-Leu16-IL-2 which retained full CD20 binding activity and showed enhanced ADCC effector function relative to a control MAb (rituximab). This novel molecule has been demonstrated to be effective in vivo in a Daudi Burkitt lymphoma model. We investigated the therapeutic potential in a rituximab resistant CD20+ human lymphoma xenograft mouse model. Materials & Methods: The mature plasmocytic B-cell line ARH77 was stably transfected with the firefly luciferase-gene using electroporation. 2x106 luciferase-transfected ARH77 cells (ARH77:luc) were inoculated s.c. into the right flank of 6–8 week old SCID mice. Groups of 8 ARH77:luc tumor bearing mice were injected i.v. with 3 doses of DI-Leu16-IL2 (25μg), Rituximab (20μg) or PBS on day 0, 2, and 4. Tumor burden was monitored by both physical measurements and bioluminescence imaging. Mice were to be sacrificed at a tumor volume > 2000mm3. Results: In this regimen DI-Leu16-IL2 treated mice demonstrated significant reduction in tumor growth whereas rituximab or the PBS control had no effect on ARH77:luc tumor progression. An increase of survival from 28 to 44 days, relative to rituximab treated mice and PBS-control, was seen. Re-growth of tumors in the DI-Leu16-IL2 group was observed after an average delay of 16 days. There were no signs of toxicity. Multiple regression analyses between tumor volume and light emission were performed which revealed a correlation between tumor mass and bioluminescence for the DI-Leu16-IL-2 group (r2=0.64), rituximab group (r2=0.61), PBS-group (r2=0.82), and the combined data sets (r2=0.7). Conclusions: DI-Leu16-IL2 demonstrated significant anti-tumor activity against rituxan-resistant human lymphoma xenografts using both physical measures and bioluminescence imaging. Despite a shorter half-life it was more effective than rituximab. Previous studies have shown the anti-tumor effects of DI-Leu16-IL2 were not entirely dependent on FcR binding suggesting that both ADCC and IL-2 targeting contribute to the superior anti-tumor activity of the immunocytokine. Ongoing studies include the use of repeat dosing cycles of this novel immunocytokine for best response using the SCID mouse/ARH77:luc tumor model. DI-Leu16-IL2 may offer promising therapeutic potential with presumed low immunogenicity for patients with CD20+ lymphoma.

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Hormaeche, Itsaso, Kim L. Rice, Arthur Zelent, Melanie J. McConnell et Jonathan D. Licht. « PLZF-RARα Utilizes the Histone Methyl Transferase G9a/GLP and the Histone Demethylase LSD1 to Repress RARα Target Genes and Block Myeloid Differentiation ». Blood 112, n^o 11 (16 novembre 2008) : 198. http://dx.doi.org/10.1182/blood.v112.11.198.198.

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Abstract As a result of the t(11;17) translocation in retinoic acid resistant subtype of acute promyelocytic leukemia (APL), the transcriptional repression domains of the Promyelocytic Leukemia Zinc Finger protein (PLZF) are fused to the ligand binding and DNA binding domains of the Retinoic Acid Receptor α (RARα). The expression of PLZF-RARα as well as the reciprocal RARα-PLZF protein both appear to contribute to leukemogenesis. While the mode of action of PML-RARα has been studied in detail, less is known about transcriptional repression mediated by PLZF-RARα. We and others previously showed an important role of histone deacetylases in PLZF and PLZF-RARα mediated gene repression. We now find that expression of PLZF-RARα also modulates gene expression through changes in the state of histone methylation at target promoters. PLZF-RARα co-precipitated in vivo with endogenous G9a, a histone methyl transferase responsible for the mono and di-methylation of euchromatic histone 3 lysine tail residue 9 (H3K9me1/2), a covalent modification associated with gene repression. Deletion analysis of the PLZF-RARα fusion protein showed that the BTB/POZ domain of PLZF fused to RARα was sufficient to mediate this interaction. PLZF-RARα also bound in vivo to LSD1, a histone demethylase that removes methyl groups from mono or di-methylated Histone 3 lysine 4 (H3K4me1/2), a change generally associated with gene repression. As with G9a the BTB/POZ domain of PLZF was implicated in binding to LSD1. Co-precipitation experiments showed a robust interaction between PLZF-RARα and G9a and LSD1 while RARα, PML-RARα and NPM-RARα bound much more weakly, suggesting that the interaction with these histone modifying enzymes may be a mechanism relatively specific to t(11;17)-associated APL. To identify genes modulated by PLZF-RARα and determine how PLZF-RARα affects the chromatin of such genes we induced expression of PLZF-RARα in a U937 tetracycline-regulated system. PLZF-RARα directly repressed known RARα target genes such as NFE2, PRAM1 and C/EBPε. As a result of PLZF-RARα expression, U937T cells were blocked in differentiation characterized by decreased expression of the myeloid cell surface markers CD11b, CD14 and CD33. Chromatin immunoprecipitation experiments in this cell line showed that PLZF-RARα expression was associated with an increase in H3K9me1/me2 at the NFE2, PRAM1 and C/EBPε promoters. Knockdown of endogenous G9a by shRNA transduction reversed transcriptional repression mediated by the fusion protein on all three promoters. Both results are consistent with the presence of G9a in PLZF-RARα transcriptional complex. By contrast, the H3K4 methylation changes in response to PLZF-RARα were promoter specific and complex: while NFE2 exhibited a decrease in H3K4me1/2, consistent with the recruitment of LSD1 and demethylation, PRAM1 and C/EBPε showed an increase in these two modifications. Inhibition of LSD1 by tranylcypromine treatment as well as knockdown of LSD1 by shRNA only reverted PLZF-RARα repression of NFE2. PLZF-RARα recruitment to all three genes was associated with a decrease in H3K4trimethylation, a modification only accomplished by jumanji-class histone demethylases. Consistent with the biochemical information, knockdown of G9a or its heterodimeric partner GLP, showed a strong biological phenotype, reverting the block in myeloid differentiation caused by PLZF-RARα as measured by the expression of the myeloid cell surface markers CD11b and CD14. Depletion of LSD1 only modestly interfered with the differentiation block mediated by the fusion protein. Gene regulation by PLZF-RARα is associated with a complex set of chromatin changes mediated by a combination of histone deacetylases, methyl transferase and demethylases. All three classes of enzymes may represent therapeutic targets in t(11;17)-APL.

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Suriano, Marco, Giovanni Martinelli, Angela Poerio, Marilina Amabile, Gianantonio Rosti, Fausto Castagnetti, Ilaria Iacobucci et al. « Evaluation of the GeneXpert Assay for the Monitoring of BCR-ABL Transcript Levels In Chronic Myeloid Leukemia (CML) Patients : Preliminary Results of a Comparison with the Manual and Traditional Manual TaqMan RQ-PCR Assay ». Blood 116, n^o 21 (19 novembre 2010) : 4831. http://dx.doi.org/10.1182/blood.v116.21.4831.4831.

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Abstract Abstract 4831 Patient with CML harbor the chromosomal translocation t(9;22) which corresponds to fusion of the BCR and ABL genes at the DNA level. Traditionally, CML disease status has been monitored by detecting the t(9;22) translocation by cytogenetics, fluorescence in situ hybridization or southern blot. To date, reverse transcriptase-polymerase chain reaction (RQ-PCR) is considered the most sensitive method available for detecting low copy numbers of the BCR-ABL gene fusion. Current RQ-PCR Taqman assay used to detect the BCR-ABL transcript are relatively time consuming because they require RNA purification and reaction setup steps. The GeneXpert BCR-ABL Monitor Assay (Cepheid, Sunnyvale, Calif) simplifies the testing procedure by using a single use cartridge, which automates the step involved and therefore significantly reduces the technical time required to run the assay. The closed system used in this assay also reduces the chance of contamination. The aim of this study was to compare the two methods of BCR-ABL quantification: the manual TaqMan RQ-PCR and the GeneXpert based assay. We analyzed 100 peripheral blood samples from CML patients with both methods. First of all, we performed a comparison in terms of quality of the ABL control gene. We found that: 87/100 (87%) samples evaluated with the TaqMan standard assay had the control gene transcript value more than 1000 copies; 100/100 (100%) samples evaluated with the GeneXpert had the control gene transcript value more than 1000 copies; Thereafter, we analyzed the concordance of the two methods in the quantification of the target gene BCR-ABL. We considered for this analysis only samples with a good quality of the ABL control gene (> 1000 copies) and we subdivided them in two groups:,: patient who achieved a major molecular response (MMR) and patients who did not achieve a MMR. The MMR wass defined as the achievement of a ratio BCR-ABL/ABLx100<0,1, according to the International Scale. We found that: 78/87 (90%) patients analyzed with the standard TaqMan assay achieved a MMR; 9/87 (10%) patients did not achieve a MMR if analyzed with the standard TaqMan protocol; 69/87 (79%) patients analyzed with the GeneXpert achieved a MMR; 18/87 (21%) patients didn't achieve a MMR if analyzed with the GeneXpert. In conclusion, our preliminary results showed that GeneXpert assay may be a valid alternative to conventional and traditional TaqMan RQ-PCR methods, allowing a better standardization of the molecular techniques used for the monitoring of minimal residual disease in CML patients. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Martinelli: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy. Rosti: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Baccarani: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria.

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VANHOOREN, Johannes C. T., Peter MARYNEN, Guy P. MANNAERTS et Paul P. VAN VELDHOVEN. « Evidence for the existence of a pristanoyl-CoA oxidase gene in man ». Biochemical Journal 325, n^o 3 (1 août 1997) : 593–99. http://dx.doi.org/10.1042/bj3250593.

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In the rat, 2-methyl branched fatty acids and the bile acid intermediates di- and tri-hydroxycoprostanic acids are desaturated by pristanoyl-CoA oxidase and trihydroxycoprostanoyl-CoA oxidase respectively. In the human, these compounds are oxidized by a single enzyme, branched-chain acyl-CoA oxidase, which according to its amino acid sequence is the human homologue of rat trihydroxycoprostanoyl-CoA oxidase. Pristanoyl-CoA oxidase is apparently absent from human tissues as indicated by immunoblot analysis [Van Veldhoven, Van Rompuy, Fransen, de Béthune and Mannaerts (1994) Eur. J. Biochem. 222, 795–801] and Northern-blot analysis [Vanhooren, Fransen, de Béthune, Baumgart, Baes, Torrekens, Van Leuven, Mannaerts and Van Veldhoven (1996) Eur. J. Biochem. 239, 302–309] of human tissues. In this paper we present evidence, however, that at least the gene for pristanoyl-CoA oxidase is present in the human. A human liver cDNA encoding a protein of 700 amino acids, showing 75% amino acid identity with rat pristanoyl-CoA oxidase and harbouring a peroxisomal C-terminal-targeting signal (SKL), was isolated. Bacterial expression of the cDNA resulted in a fusion protein that was cross-reactive with antibodies directed against rat pristanoyl-CoA oxidase and the C-terminal SKL sequence. Screening of a genomic library with the isolated cDNA as a probe resulted in a genomic clone in which four introns were localized. By means of fluorescence in situhybridization the gene for human pristanoyl-CoA oxidase was mapped at chromosome position 4p15.3. We conclude that a gene for pristanoyl-CoA oxidase is present in the human genome. The gene appears to be expressed to such a low extent in liver that its mRNA cannot be detected by routine Northern-blot analysis and that its product remains undetected by standard immunoblotting or by enzyme activity measurements. We speculate that the gene may be expressed under special (e.g. certain developmental stages) conditions or in certain specialized tissues not examined thus far.

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TAMBURRO, Antonio, Nerino ALLOCATI, Michele MASULLI, Domenico ROTILIO, Carmine DI ILIO et Bartolo FAVALORO. « Bacterial peptide methionine sulphoxide reductase : co-induction with glutathione S-transferase during chemical stress conditions ». Biochemical Journal 360, n^o 3 (10 décembre 2001) : 675–81. http://dx.doi.org/10.1042/bj3600675.

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Peptide methionine sulphoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous enzyme catalysing the reduction of methionine sulphoxide to methionine in proteins, while the glutathione S-transferases (GSTs) are a major family of detoxification enzymes. A gene homologous to MsrA was identified in a chromosomal fragment from the bacterium Ochrobactrum anthropi, and this gene is located just downstream of a GST gene identified previously (OaGST) [Favaloro, Tamburro, Angelucci, De Luca, Melino, Di Ilio and Rotilio (1998) Biochem. J. 335, 573–579]. This raises the question of whether the products of these two genes may be involved in a common cellular protection function. To test this hypothesis, the hypothetical MsrA protein has been overexpressed in Escherichia coli as a functional 51kDa GST fusion protein. Following cleavage with thrombin and purification, the soluble 24kDa protein showed MsrA activity with N-acetylmethionine sulphoxide as substrate, as well as with other sulphoxide compounds. Therefore polyclonal antibodies were raised against the recombinant protein, and the modulation of MsrA in this bacterium, grown in the presence of different stimulants simulating several stress conditions, was investigated. The level of expression of MsrA was detected both by measuring the mRNA level and by immunoblotting experiments, in addition to measuring its catalytic activity. MsrA is a constitutive enzyme which is also inducible by chemical stress involving phenolic compounds such as phenol and 4-chlorophenol. Recently we reported that the GST of this bacterium, like MsrA, is only modulated by toxic chemical compounds [Favaloro, Tamburro, Trofino, Bologna, Rotilio and Heipieper (2000) Biochem. J. 346, 553–559]; therefore this is the first indication of a co-induction of the MsrA and GST enzymes during chemical stress.

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Huang, Bixing, Cynthia B. Whitchurch et John S. Mattick. « FimX, a Multidomain Protein Connecting EnvironmentalSignals to Twitching Motility in Pseudomonasaeruginosa ». Journal of Bacteriology 185, n^o 24 (15 décembre 2003) : 7068–76. http://dx.doi.org/10.1128/jb.185.24.7068-7076.2003.

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ABSTRACT Twitching motility is a form of surface translocation mediated by the extension, tethering, and retraction of type IV pili. Three independent Tn5-B21 mutations of Pseudomonas aeruginosa with reduced twitching motility were identified in a new locus which encodes a predicted protein of unknown function annotated PA4959 in the P. aeruginosa genome sequence. Complementation of these mutants with the wild-type PA4959 gene, which we designated fimX, restored normal twitching motility. fimX mutants were found to express normal levels of pilin and remained sensitive to pilus-specific bacteriophages, but they exhibited very low levels of surface pili, suggesting that normal pilus function was impaired. The fimX gene product has a molecular weight of 76,000 and contains four predicted domains that are commonly found in signal transduction proteins: a putative response regulator (CheY-like) domain, a PAS-PAC domain (commonly involved in environmental sensing), and DUF1 (or GGDEF) and DUF2 (or EAL) domains, which are thought to be involved in cyclic di-GMP metabolism. Red fluorescent protein fusion experiments showed that FimX is located at one pole of the cell via sequences adjacent to its CheY-like domain. Twitching motility in fimX mutants was found to respond relatively normally to a range of environmental factors but could not be stimulated by tryptone and mucin. These data suggest that fimX is involved in the regulation of twitching motility in response to environmental cues.

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Putra, IGAA, AAAM Adi et NM Astawa. « Variasi Genetik Gen Penyandi Protein Fusi dari Avian Paramyxovirus Tipe I di Bali (GENETIC VARIATION OF GENE ENCODING FUSION PROTEIN OF AVIAN PARAMYXOVIRUS TYPE-I IN BALI) ». Jurnal Veteriner 17, n^o 2 (15 juillet 2016) : 211–17. http://dx.doi.org/10.19087/jveteriner.2016.17.2.211.

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Xu, Jie, Wu Zhang, Xiaojing Yan, Chen Zhao, Jiang Zhu, Zhu Chen, Sai-Juan Chen et Jiong Hu. « NPM1 Mutation Contributes to Hematological Dysfunction By Disrupting H3K79 Methylation ». Blood 128, n^o 22 (2 décembre 2016) : 2702. http://dx.doi.org/10.1182/blood.v128.22.2702.2702.

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Abstract NPM1 is one of the most frequent acquired mutated genes in acute myeloid leukemia (AML). Previous studies have shown that NPM1 mutation (NPMc+) established the distinctive gene expression signatures, which were associated with mixed lineage leukemia (MLL)-target genes, like MEIS1 and HOXA cluster. In AML carrying MLL fusion-oncoproteins, DOT1L-mediated histone 3 lysine 79 (H3K79) methylation is implicated in the regulation of MLL-target genes. Compared with MLL abnormalities, NPM1 variants preserve the similar transcriptional characteristics. However, whether NPM1 mutation could affect the histone modification of H3K79 methylation is unknown. In this study, we showed that NPM1 mutation dysregulated the homeostasis of hematopoietic stem and progenitor cells and resulted in ageing-related myeloproliferation in NPMc+ transgenic mouse model. Interestingly, through scanning the chromatin modification related gene profiling, di- and tri- methylated H3K79 were significantly elevated in bone marrow (BM) Lin-Sca-1+c-Kit+ cells (LSKs) of NPMc+ mice comparing to wild type (WT). Meanwhile, in the leukemia cell lines and AML primary BM samples, we confirmed that NPM1 mutated cells expressed the higher level of H3K79 methylation. In vitro assays also indicated that the decrease or increase of methylated H3K79 could be regulated respectively by knockdown or overexpression of NPM1 mutant but not WT. Importantly, with DOT1L inhibitor treatment, reduced di- and tri- methylated H3K79 was observed in OCI-AML3 (NPMc+) strains but not OCI-AML2 (NPM1 WT) cells. In contrast with OCI-AML2, DOT1L inhibitor significantly promoted the cell apoptosis and restrained the cell cycle of OCI-AML3. Moreover, by the means of murine BM colony formation assay, DOT1L inhibitor obviously weakened myeloid cell proliferation in NPMc+ mice, while colony number in WT group did not change. Also, leukemia development was repressed in OCI-AML3-xenografted NOD/SCID mice with the treatment of DOT1L inhibitor. Taken together, NPM1 mutation contributes to hematological dysfunction by disrupting H3K79 methylation, which could be largely attenuated by DOT1L inhibitor. Disclosures No relevant conflicts of interest to declare.

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Mach, Robert L., Clemens K. Peterbauer, Kathrin Payer, Sylvia Jaksits, Sheridan L. Woo, Susanne Zeilinger, Cornelia M. Kullnig, Matteo Lorito et Christian P. Kubicek. « Expression of Two Major Chitinase Genes of Trichoderma atroviride (T. harzianum P1) Is Triggered by Different Regulatory Signals ». Applied and Environmental Microbiology 65, n^o 5 (1 mai 1999) : 1858–63. http://dx.doi.org/10.1128/aem.65.5.1858-1863.1999.

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ABSTRACT Regulation of the expression of the two major chitinase genes,ech42 (encoding the CHIT42 endochitinase) andnag1 (encoding the CHIT73N-acetyl-β-d-glucosaminidase), of the chitinolytic system of the mycoparasitic biocontrol fungusTrichoderma atroviride (= Trichoderma harzianumP1) was investigated by using a reporter system based on theAspergillus niger glucose oxidase. Strains harboring fusions of the ech42 or nag1 5′ upstream noncoding sequences with the A. niger goxAgene displayed a glucose oxidase activity pattern that was consistent under various conditions with expression of the nativeech42 and nag1 genes, as assayed by Northern analysis. The expression product of goxA in the mutants was completely secreted into the medium, detectable on Western blots, and quantifiable by enzyme-linked immunosorbent assay. nag1gene expression was triggered during growth on fungal (Botrytis cinerea) cell walls and on the chitin degradation productN-acetylglucosamine. N-Acetylglucosamine, di-N-acetylchitobiose, or tri-N-acetylchitotriose also induced nag1 gene expression when added to mycelia pregrown on different carbon sources.ech42 expression was also observed during growth on fungal cell walls but, in contrast, was not triggered by addition of chitooligomers to pregrown mycelia. Significant ech42expression was observed after prolonged carbon starvation, independent of the use of glucose or glycerol as a carbon source, suggesting that relief of carbon catabolite repression was not involved in induction during starvation. In addition, ech42 gene transcription was triggered by physiological stress, such as low temperature, high osmotic pressure, or the addition of ethanol. Four copies of a putative stress response element (CCCCT) were found in the ech42promoter.

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Rejlova, Katerina, Karolina Kramarzova, Meritxell Alberich-Jorda, Karel Fiser, Marketa Zaliova, Jan Trka et Julia Starkova. « The Role of Histone Demethylases in the Transcription Regulation of HOX Genes in PML-RARa+ AML Patients ». Blood 124, n^o 21 (6 décembre 2014) : 876. http://dx.doi.org/10.1182/blood.v124.21.876.876.

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Abstract Homeobox genes (HOX) encode transcription factors that are frequently deregulated in leukemias. Our previous findings described that HOX gene expression differs among genetically characterized subtypes of pediatric AML with PML-RARa+ patients having the lowest overall HOX gene expression. We observed that HOX gene expression positively correlated with expression of histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX and negatively with DNA methyltransferase DNMT3b. Interestingly, it has been shown that JMJD3 is a direct target of PML-RARa protein (Martens, JH et al, 2010, Cancer Cell). These findings led us to postulate the hypothesis that reduced levels of HOX genes in PML-RARa+ AML can be caused by the suppressed expression of histone demethylases, such as JMJD3 and UTX, resulting in increased H3K27 methylation and transcription inhibition. We chose PML-RARa+ NB4 cell line to study the role of PML-RARa fusion gene in the regulation of HOX gene expression. To inhibit the effect of PML-RARa we used all-trans retinoic acid (ATRA; 1 uM, 10 uM) which was described to release the block caused by this fusion protein. Expression of particular HOX genes (e.g., HOXA1, HOXA3, HOXA5, HOXA7) together with that of JMJD3 and UTX assessed by qPCR was significantly elevated after ATRA treatment, while gene expression of DNMT3b was decreased. To test whether the reduction in HOX gene expression is directly related to the levels of JMJD3 and UTX, we cultured NB4 cells with a specific inhibitor of these histone demethylases, GSK-J4 (1 uM, 10 uM), in combination with ATRA. This co-treatment led to inhibition of JMJD3 and UTX proteins, followed by significant reduction of HOX genes expression (e.g., HOXA1, HOXA3, HOXA5, HOXA7). This result supports our hypothesis that HOX genes expression is directly related to JMJD3/UTX activity. To determine the effect of ATRA and GSK-J4 on histone marks we have isolated histones by acid extraction and detected the levels of histones by western blot in NB4 ATRA or GSK-J4/ATRA treated cells. We observed that the level of repressive histone methylation mark (trimethylated H3K27; H3K27me3) was decreased after ATRA treatment (activation of JMJD3/UTX) and increased after GSK-J4/ATRA co-treatment (inhibition of JMJD3/UTX). The opposite effect was observed in active histone methylation marks where di- and tri-methylated H3K4 (H3K4me2, H3K4me3) increased after ATRA treatment and decreased after GSK-J4/ATRA co-treatment. H3K9 dimethylated (another repressive histone methylation mark) levels did not change. Next, to investigate the histone code directly in particular HOX genes regions we performed chromatin immunoprecipitation (ChIP) assays. We studied the presence of H3K27me3 and H3K4me2 in 5´UTR genomic region of particular HOX genes (HOXA1, HOXA2, HOXA3, HOXA5, HOXA7) in cells treated with ATRA alone or in the combination with GSK-J4. Preliminary results showed reduction in repressive marks (H3K27me3) upon ATRA treatment, whereas addition of GSK-J4 prevented this decrease. Accordingly, we observed that ATRA/GSK-J4 co-treatment reduced active histone mark H3K4me2. To evaluate the role of DNA methylation in observed expression changes after ATRA treatment we performed bisulfite sequencing of particular promoter sites of HOX genes (e.g., HOXA7, HOXA5). Although we detected decreased DNMT3b gene expression after ATRA treatment there was no change in DNA methylation of CpGs in studied regions. Our results demonstrate that changes in chromatin activity correspond with changes in HOX gene expression. Moreover, ChIP data show direct binding of the modified histones and HOX 5´UTR sites. Our data implicate histone demethylases in regulation of HOX gene expression in PML-RARa+ leukemic blasts. DNA methylation in these particular HOX genes is not involved in the regulation. Elucidating the mechanism of regulation of HOX genes expression can help to understand their role in the leukemogenic process. Supported by GACR P304/12/2214 and GAUK 568213. Disclosures No relevant conflicts of interest to declare.

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David Chen, Chun-Wei, Amit U. Sinha, Jun Qi, Aniruddha J. Deshpande, Nan Zhu, Richard Koche, Rowena Eng et al. « Genome-Wide RNAi Screen Identifies The Mechanistic Role For DOT1L In MLL-Rearranged Leukemia ». Blood 122, n^o 21 (15 novembre 2013) : 598. http://dx.doi.org/10.1182/blood.v122.21.598.598.

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Abstract Rearrangement of Mixed-Lineage Leukemia (MLL) gene defines a genetically distinguishable subset of aggressive leukemias with poor prognosis. Recent studies exhibit promising activity of small-molecule inhibitors of the H3K79 methyltransferase DOT1L (disruptor of telomeric silencing-1 like) against leukemias bearing MLL-translocations (Daigle et. al. 2011 Cancer Cell). However, the mechanisms underlying the epigenetic addiction of MLL-fusion oncogenic program to H3K79-methylation remain unclear. A number of labs have recently shown the expression of MLL-fusion target genes including HOXA cluster and MEIS1 is strongly dependent on DOT1L activity, whereas most of the normal genes do not require H3K79 methylation for their expression. To further investigate this unique subordination of MLL-fusion leukemic program to DOT1L/H3K79-methylation, we sought to identify genes, whose suppression would rescue the Dot1l loss-of-function phenotype in mouse MLL-AF9 cells. We conducted a suppressor screen by introducing a mouse genome-wide shRNA library (92,425 shRNA targeting 16,469 mouse genes) into Dot1lf/f-MLL-AF9 leukemic cells harboring tamoxifen-inducible Cre recombinase. Relative frequencies of integrated shRNA sequences were assessed by high throughput sequencing. Our results revealed that knockdown of sirtuin 1 (Sirt1), a histone deacetylase, significantly antagonizes the lethality cause by loss of Dot1l/H3K79me2 in MLL-AF9 leukemia. To understand the function of Sirt1 in MLL-rearranged leukemia, we performed genome-wide expression profiling and revealed that Sirt1 is essential for complete silencing of the MLL-AF9 driven oncogenic program including Hoxa7 and Meis1 upon inhibition of Dot1l. ChIP-seq analyses indicate that Sirt1 localizes to MLL-AF9 targets and mediates loss of H3K9ac upon suppression of Dot1l. Because Sirt1 is known to interact with epigenetic repressive enzymes such as polycomb repressive complex (PRC) members and Suv39h1, we them examine whether Sirt1 participates in suppression of MLL-AF9 targets by recruiting these silencing mechanisms to the loci. ChIP-seq analyses showed that suppression of Dot1l activity resulted in increased H3K9me2 and H3K27me3 levels near the transcriptional start site of MLL-AF9 target genes. Remarkably, knockdown of Sirt1 blocks the accumulation of H3K9me2 and maintains the chromatin accessibility at MLL-AF9 targets after Dot1l suppression, whereas the increase of H3K27me3 at those loci was minimally affected. Additionally, we identified that histone methyltransferase Suv39h1, a well characterized Sirt1 binding partner, is responsible for H3K9 di-methylation and gene silencing of Hoxa7 and Meis1 in response to DOT1L inhibitor. Finally, we showed that pharmacological activation of Sirt1 and inhibition of Dot1l synergistically suppresses the leukemogenetic potential of MLL-AF9 cells in a secondary leukemia drug exposure model. Our results suggest that aberrant methylation of H3K79 may render leukemic transformation by inhibition of repressive mechanisms (including SIRT1/SUV39H1-mediated heterochromatin formation and others) at MLL-fusion targets. Combination treatment model further implies that patients with leukemia bearing MLL-translocations may benefit from synergistic therapies that simultaneously target both DOT1L and SIRT1 or other epigenetic regulators. Disclosures: No relevant conflicts of interest to declare.

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Bearden, Scott W., Teanna M. Staggs et Robert D. Perry. « An ABC Transporter System of Yersinia pestis Allows Utilization of Chelated Iron by Escherichia coliSAB11 ». Journal of Bacteriology 180, n^o 5 (1 mars 1998) : 1135–47. http://dx.doi.org/10.1128/jb.180.5.1135-1147.1998.

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ABSTRACT The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA ofY. pestis KIM6+ was used for transduction of anEscherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The ∼21-kDa protein encoded by the separately transcribedyfeE gene may be located in the cell envelope, since ayfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media.

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Kansal, Rita, David A. Rasko, Jason W. Sahl, George P. Munson, Koushik Roy, Qingwei Luo, Alaullah Sheikh, Kurt J. Kuhne et James M. Fleckenstein. « Transcriptional Modulation of Enterotoxigenic Escherichia coli Virulence Genes in Response to Epithelial Cell Interactions ». Infection and Immunity 81, n^o 1 (31 octobre 2012) : 259–70. http://dx.doi.org/10.1128/iai.00919-12.

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EnterotoxigenicEscherichia coli(ETEC) strains are a leading cause of morbidity and mortality due to diarrheal illness in developing countries. There is currently no effective vaccine against these important pathogens. Because genes modulated by pathogen-host interactions potentially encode putative vaccine targets, we investigated changes in gene expression and surface morphology of ETEC upon interaction with intestinal epithelial cellsin vitro. Pan-genome microarrays, quantitative reverse transcriptase PCR (qRT-PCR), and transcriptional reporter fusions of selected promoters were used to study changes in ETEC transcriptomes. Flow cytometry, immunofluorescence microscopy, and scanning electron microscopy were used to investigate alterations in surface antigen expression and morphology following pathogen-host interactions. Following host cell contact, genes for motility, adhesion, toxin production, immunodominant peptides, and key regulatory molecules, including cyclic AMP (cAMP) receptor protein (CRP) and c-di-GMP, were substantially modulated. These changes were accompanied by visible changes in both ETEC architecture and the expression of surface antigens, including a novel highly conserved adhesin molecule, EaeH. The studies reported here suggest that pathogen-host interactions are finely orchestrated by ETEC and are characterized by coordinated responses involving the sequential deployment of multiple virulence molecules. Elucidation of the molecular details of these interactions could highlight novel strategies for development of vaccines for these important pathogens.

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Jo, Stephanie Y., Eric M. Granowicz et Jay L. Hess. « Assessment of DOT1L as a Therapeutic Target In Acute Leukemia ». Blood 116, n^o 21 (19 novembre 2010) : 3291. http://dx.doi.org/10.1182/blood.v116.21.3291.3291.

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Abstract Abstract 3291 Histone modifying enzymes are crucial regulators of hematopoiesis that are commonly disrupted in acute leukemia. DOT1L has emerged as a particularly important methyltransferase in leukemias with Mixed Lineage Leukemia (MLL) rearrangements. Leukemogenic MLL fusion proteins transform primarily through upregulation of A-cluster HOX genes, including HOXA9 and the HOX cofactor MEIS1. Many of the most common MLL translocation partners including the AF4 family members, AF9, ENL, and AF10, form the Elongation Assisting Proteins (EAP) complex that includes DOT1L. DOT1L is the only known histone methyltransferase that methylates histone H3 on lysine 79 (H3K79). Increasing evidence suggests this histone modification, which is generally associated with transcriptional activation, is essential for MLL fusion protein mediated oncogenicity. Chromatin immunoprecipitation (ChIP) on MLL fusion protein containing cell lines shows higher levels of H3K79 methylation across the HOXA9 and MEIS1 loci compared to non-MLL fusion protein containing cell lines. Similarly, patient samples with MLL fusion proteins show elevated H3K79 methylation. Finally, knockdown of DOT1L has been shown to inhibit growth of MLL rearranged cell lines. These findings suggest that DOT1L may be an effective therapeutic target, however further development of DOT1L inhibitors will be dependent on assessing the efficacy of DOT1L disruption in a wider range of leukemic cells as well as determining the potential toxicity and effect on normal hematopoiesis. Given that constitutive Dot1l knock out is early embryonic lethal, we established conditional Dot1l knockout mouse from gene trap sperm obtained from the Knock Out Mouse Project (KOMP). Dot1l targeted animals were crossed to CreER animals so that Dot1l knock out could be induced with 4-OHT or tamoxifen. To determine the effects of Dot1l deletion in transformation ability, mouse were injected with 5-flurouracil and bone marrow cells were transduced with retrovirus expressing oncogenes in the presence or absence of 4-OHT and growth in methocult media was examined. These experiments showed that growth of cells transformed by MLL-AF9 is completely abolished by Dot1l deletion while transformations by HOXA9/MEIS1 (downstream targets of MLL fusion proteins) and E2A-HLF (which expresses very low levels of HOXA9/MEIS1 and transform through other mechanisms) were unaffected. We also examined the toxicity of Dot1l deletion in vivo by treating mice with tamoxifen and monitoring survival and assessing hematopoiesis. Immunohistochemical studies show that loss of Dot1l is associated with widespread loss of lysine 79 di- and tri-methylation in organs including liver, spleen, bone marrow, testis, muscle and gastrointestinal track with some residual methylation retained in the central nervous system. Immunophenotypic analysis of mouse bone marrow 3–4 weeks after tamoxifen treatment revealed reduction in HSCs, GMPs, MEPs, and CLPs with Dot1l excision. Bone marrow transplantations and cell cycle experiments are currently underway to further characterize the hematopoietic defects in Dot1l deficient animals. Additional experiments will be needed to determine if Dot1l activity is required in other leukemias with high level HOX expression. Together these results suggest that Dot1l is a promising therapeutic target as it is specifically required for transformation by MLL fusion leukemia, however bone marrow suppression occurs with Dot1l inhibition. Disclosures: No relevant conflicts of interest to declare.

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Grantcharova, Nina, Verena Peters, Claudia Monteiro, Katherina Zakikhany et Ute Römling. « Bistable Expression of CsgD in Biofilm Development of Salmonella enterica Serovar Typhimurium ». Journal of Bacteriology 192, n^o 2 (6 novembre 2009) : 456–66. http://dx.doi.org/10.1128/jb.01826-08.

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ABSTRACT Bacterial persistence in the environment and in the infected host is often aided by the formation of exopolymer-enclosed communities known as biofilms. Heterogeneous gene expression takes place in microcompartments formed within the complex biofilm structure. This study describes cell differentiation within an isogenic bacterial cell population based on the example of biofilm formation by Salmonella enterica serovar Typhimurium. We analyzed the expression of the major biofilm regulator CsgD at the single-cell level with a chromosomal CsgD-green fluorescent protein (GFP) translational fusion. In individual cells, CsgD-GFP expression is mostly found in the cytoplasm. Quantitative expression analysis and results from three different models of S. Typhimurium biofilms demonstrated that CsgD is expressed in a bistable manner during biofilm development. CsgD expression is, however, monomodal when CsgD is expressed in larger amounts due to a promoter mutation or elevated levels of the secondary signaling molecule c-di-GMP. High levels of CsgD-GFP are associated with cellular aggregation in all three biofilm models. Furthermore, the subpopulation of cells expressing large amounts of CsgD is engaged in cellulose production during red, dry, and rough (rdar) morphotype development and in microcolony formation under conditions of continuous flow. Consequently, bistability at the level of CsgD expression leads to a corresponding pattern of task distribution in S. Typhimurium biofilms.

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Villiers, William, Paul Lavender, Audrey Kelly, Michael Lim, Cameron Osborne et Richard Dillon. « Untangling the Transcriptional Mis-Regulation Driven By Pml;rara ». Blood 134, Supplement_1 (13 novembre 2019) : 282. http://dx.doi.org/10.1182/blood-2019-125555.

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Background:The PML;RARA fusion protein is the hallmark driver of Acute Promyelocytic Leukemia (APL). The fusion disrupts retinoic acid signalling, leading to the proliferation of myeloid precursors halted at the promyelocyte stage of maturation. Chromatin conformation plays a fundamental role in controlling regulatory networks driving cell differentiation, however the genome wide organisational changes that occur during the PML;RARA differentiation block remains to be elucidated. Aims: We have aimed to characterise three hallmarks of PML;RARA driven transcriptional mis-regulation: transcription factor binding, epigenetic remodelling and higher order chromatin organisation. Methods: To a PML;RARA inducible U937-PR9 cell line model (Figure 1a), we have applied 1) Promoter Capture Hi-C (PCHi-C) to characterise long-range regulatory interactions, 2) Cut&Run, a newly described chromatin profiling technique, and 3) RNA-seq. All datasets are paired and have been modelled as induced vs non-induced. Results: PML;RARA induction resulted in 855 significantly differentially expressed genes (DEGs, adjusted pvalue < 0.01). Up regulated genes (457) were enriched for genes involved in proliferative and oncogenic pathways. Down regulated genes (398) were enriched for drivers of cell differentiation, including the master regulators of myeloid differentiation: CEBPE, CEBPB and CEBPA. We identified 15,412 PML;RARA binding sites using Cut&Run (n=2). These sites encompassed 95% of previously uncovered PML;RARA binding sites, in addition to ~12,000 novel sites. 53% of these regions were distributed at gene promoters, 23% were intergenic and 24% at gene bodies. We observed a global decrease in H3k27ac after PML;RARA induction (86% regional loss), a significant proportion of these regions coincide with PML;RARA binding (Fisher's test 2.2-16). 1,900 PML;RARA peaks were associated with 66% DEGs. Interestingly, a significant proportion of PML;RARA peaks were not associated with DEGs. Applying PCHi-C (n=3) we identified >60,000 consistent differential interactions (DIs, ihw < 0.01), with a mean interaction distance of 100kb. 30,039 interactions increased and 30,403 interactions decreased after PML;RARA induction. Over half of DIs directly involved 8,066 PML;RARA binding sites (Fisher's test 2.2-16). We observed that genes losing long-range interactions were more likely to have decreased expression (59%) and this observation increased if a gene is also bound by PML;RARA (65%). SPI1, ID2, CEBPA, CEBPB, CEBPE, EGR1, and TFEB, key regulators of myeloid differentiation, display this pattern of PML;RARA driven negative regulation. We also observed that genes with increased expression were more likely to gain long-range interactions (60%). The top ranked gene across all datasets was the prostaglandin receptor 4 (PTGER4), a G-Protein coupled receptor highly expressed in AML and upregulated in our datasets (3.1 fold) (figure 1b). PTGER4 gained long-range interactions with an intergenic region ~300kb downstream of its promoter. This intergenic region is highly enriched for both H3k27ac and PML;RARA. This suggests that PML;RARA facilitates the long-range activation of PTGER4. We see a similar pattern to PTGER4 in 15% of DEGs where PML;RARA is anchored to both ends of the DI. Summary/Conclusion: Applying novel NGS techniques to a simple model, we have highlighted that specific chromosome conformations are pivotal to the transcriptional mis-regulation driven by PML;RARA. We show PML;RARA may be directly involved in the re-organisation of the genome and this 3D architecture is pivotal in driving the Leukemia differentiation block. Disclosures Dillon: Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; TEVA: Consultancy, Honoraria.

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Iacobucci, Ilaria, Emanuela Ottaviani, Federica Salmi, Viviana Guadagnuolo, Nicoletta Testoni, Annalisa Lonetti, Cristina Papayannidis et al. « Genome-Wide Analysis by High-Resolution SNP Array Identifies Novel Genomic Alterations in Acute Promyelocytic Leukemia (APL). » Blood 114, n^o 22 (20 novembre 2009) : 167. http://dx.doi.org/10.1182/blood.v114.22.167.167.

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Abstract Abstract 167 Introduction: Acute promyelocytic leukemia (APL) is characterized by the chromosomal translocation t(15;17), resulting in the fusion of the promyelocytic leukemia (PML) gene and retinoic acid receptor α (RARA) gene (PML-RARA). Experimental evidence obtained in transgenic mice revealed that PML-RARA is necessary but not sufficient for the development of APL, suggesting that additional genetic mutations are also required for the development of APL. Aim: To define whether additional submicroscopic genomic alterations may characterize APL and be used to better classify the disease by dissection of genomic subsets. Methods: At the time of writing, DNA from the bone marrow of 21 cases of t(15;17) APL at diagnosis were examined. Genomic DNA was isolated from mononuclear APL cells and applied to Genome-Wide Human SNP 6.0 array microarrays (Affymetrix, Santa Clara, CA) following the manufacturer's instructions. Copy number aberrations were scored using the Hidden Markov Model and the segmentation approach available within the Partek software package as well as by visual inspection. All aberrations were calculated with respect to a set of 270 Hapmap normal individuals and a set of samples obtained from acute leukaemia cases in remission in order to reduce the noise of raw copy number data. When available, in order to exclude inherited copy number variants a comparison to paired constitutional DNA and to paired remission DNA was performed. Fluorescence in situ hybridization, quantitative PCR and nucleotide sequencing were used to confirm genomic alterations. Results: In all patients we identified one or more genomic abnormalities ranging from loss or gain of complete chromosome arms (trisomy 8, loss of 20q and loss of 6q) to submicroscopic genomic intervals. Focal genetic alterations were detected at the breakpoints of t(15;17)(q22;q21) in PML and RARA genes. Hemizygous deletions were identified at 1p13.3 affecting the glutathione S-transferase mu 1 (GSTM1), at 2q33.3–q34 involving ERBB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4 avian), at 3p11.2 (EPHA3), at 12p13 (ETV6), and at 17q12 (NF1). Deletions also affected genes involved in cell regulations as CDKN2A (9p21) and RB1 (13q14.2). Most frequent gains affected the TP73 gene at 1p36.3, the oncogene MYC at 8q24 (4.33 Mb), the oncogene ETS1 (11q23.3), the RAS p21 protein activator 3 (RASA3) at 13q34 and PRAME at the 22q11.22. Interestingly, in 2 cases we observed a gain of the region (5p15.33) containing the telomerase reverse transcriptase (TERT) which is important for maintenance of telomere ends. Other recurring genetic lesions were uncommon and were identified only in single cases. Some lesions affected regions lacking annotated genes and they are under investigation for miRNAs. For each alteration we interrogated a collated library of copy-number variants (CNVs, Database of Genomics Variants and USCS Genome Browser) to assure that these regions were not known as CNVs. Conclusion: These data demonstrate that different cooperating events may be involved in the generation of APL. These novel findings may be used to stratify patients according to genomic changes and to facilitate the screening for novel therapeutic targets. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, PIO project 2007, Strategico di Ateneo. Disclosures: No relevant conflicts of interest to declare.

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Stiner, Lawrence, et Larry J. Halverson. « Development and Characterization of a Green Fluorescent Protein-Based Bacterial Biosensor for Bioavailable Toluene and Related Compounds ». Applied and Environmental Microbiology 68, n^o 4 (avril 2002) : 1962–71. http://dx.doi.org/10.1128/aem.68.4.1962-1971.2002.

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ABSTRACT A green fluorescent protein-based Pseudomonas fluorescens strain A506 biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions. The biosensor is based on a plasmid carrying the toluene-benzene utilization (tbu) pathway transcriptional activator TbuT from Ralstonia pickettii PKO1 and a transcriptional fusion of its promoter PtbuA1 with a promoterless gfp gene on a broad-host-range promoter probe vector. TbuT was not limiting, since it was constitutively expressed by being fused to the neomycin phosphotransferase (nptII) promoter. The biosensor cells were readily induced, and fluorescence emission after induction periods of 3 h correlated well with toluene, benzene, ethylbenzene, and trichloroethylene concentrations. Our experiments using flow cytometry show that intermediate levels of gfp expression in response to toluene reflect uniform induction of cells. As the toluene concentration increases, the level of gfp expression per cell increases until saturation kinetics of the TbuT-PtbuA1 system are observed. Each inducer had a unique minimum concentration that was necessary for induction, with K app values that ranged from 3.3 ± 1.8 μM for toluene to 35.6 ± 16.6 μM for trichloroethylene (means ± standard errors of the means), and maximal fluorescence response. The fluorescence response was specific for alkyl-substituted benzene derivatives and branched alkenes (di- and trichloroethylene, 2-methyl-2-butene). The biosensor responded in an additive fashion to the presence of multiple inducers and was unaffected by the presence of compounds that were not inducers, such as those present in gasoline. Flow cytometry revealed that, in response to toxic concentrations of gasoline, there was a small uninduced population and another larger fully induced population whose levels of fluorescence corresponded to the amount of effectors present in the sample. These results demonstrate the potential for green fluorescent protein-based bacterial biosensors to measure environmental contaminants.

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Hazim, Antonious Z., Gordon Ruan, Aldo A. Acosta-Medina, Jithma P. Abeykoon, Aishwarya Ravindran, Robert Vassallo, Jay Ryu et al. « Classical and Non-Classical Phenotypes of Erdheim-Chester Disease : Correlating Clinical, Radiographic, and Genotypic Findings ». Blood 138, Supplement 1 (5 novembre 2021) : 2566. http://dx.doi.org/10.1182/blood-2021-144786.

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Abstract Background: Erdheim-Chester disease (ECD) is characterized by multi-organ infiltration of clonal histiocytes bearing activating mutations predominantly in the MAPK pathway. The diagnosis of ECD is clinico-pathologic; histopathologic findings alone are often non-specific. Characteristic pathognomonic finding of ECD is the symmetric osteosclerosis of the distal femur and proximal tibia/fibula, seen in >90% of cases, and referred to herein as classic ECD (C-ECD). There is a paucity of data on the phenotypic and mutational differences between C-ECD and non-classic ECD (NC-ECD). Determining phenotypic patterns may allow for earlier suspicion and diagnosis. Methods: Patients who met the revised ECD criteria proposed by Haroche J et al (Blood 2020;135:1311-1318) and had full body imaging that included the lower legs (18-FDG-PET/CT or CT/bone scan) were included. ECD diagnosis was made when >1 major criteria plus >1 minor criteria were present. Major criteria: 1) symmetric meta-diaphyseal osteosclerosis in legs; 2) "hairy kidneys"; "coated aorta", right atrial pseudotumor, xanthelasma, exophthalmos; or osteosclerosis of paranasal sinuses. Minor criteria: 1) histologic finding of typical foamy histiocytes (CD68+/CD163+/CD1a-) associated with fibrosis; 2) mutation/gene fusion of BRAF, CSF1R, or MAPK/PI3K pathways. We compared the organ involvement and BRAF V600Emutational status between C-ECD and NC-ECD. Results: A total of 105 patients were included. The median age at diagnosis was 57 years (range, 38-81) and most were males (62%). Majority had 18-FDG-PET/CT (83%) and BRAF V600E testing (65%). The main organ systems involved were skeletal (83%), renal (64%), adrenal (44%), and pulmonary (42%). Central diabetes insipidus (DI), "hairy kidneys", and "coated aorta" were present in 27 (26%), 54 (51%), and 44 (42%) patients, respectively. Among those tested, BRAF V600E mutation was found in 48/67 (72%) by immunohistochemistry. In our cohort, most patients (n=87. 83%) had C-ECD. NC-ECD had significantly lower number of organs/systems involved compared with C-ECD (median 3 vs 6, p=0.002). C-ECD had significantly higher rates of involvement of paranasal sinuses (51%/7%, p=0.002), DI (26%/0%, p=0.02), and similar rates of lung (44%/43%), cardiac (34%/14%), and skin (14%14%) involvement when compared to NC-ECD. BRAF V600E was significantly more common in C-ECD (88%/30%, p=0.004). Thirty-nine (37%) patients underwent next generation sequencing, of whom 33 (31%) had successful testing. In C-ECD, 3 patients had mutations other than BRAF V600E, these included: NRAS, MAP2K1, and MEF2C-FLT3 fusion. In NC-ECD, 5 patients had mutations other than BRAF V600E, these included: MAP2K1, KRAS, and NF1. Conclusions: Our study suggests distinct differences in clinical presentation and molecular findings exist between C-ECD and NC-ECD. C-ECD has a higher degree of organ involvement and harbor BRAF V600E more frequently than NC-ECD. Further analysis of histopathologic findings and outcomes in this cohort may provide insights into these ECD subsets that can optimize future management of this disease. Figure 1: Sites of involvement of classical Erdheim-Chester Disease (C-ECD) versus non-classical Erdheim-Chester Disease (NC-ECD) Figure 1 Figure 1. Disclosures Vassallo: Bristol-Myers-Squibb: Research Funding; Sun Pharma.: Research Funding; Pfizer: Research Funding. Tobin: Mayo Clinic Center for MS and Autoimmune Neurology: Research Funding; Mallinckrodt Pharmaceuticals: Research Funding; National Institutes of Health: Research Funding. Bennani: Verastem: Other: Advisory Board; Purdue Pharma: Other: Advisory Board; Vividion: Consultancy, Other: Advisory Board; Daichii Sankyo Inc: Other: Advisory Board; Kyowa Kirin: Other: Advisory Board.

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Reisenauer, Mary Rose, Steven W. Wang, Yang Xia et Wenzheng Zhang. « Dot1a contains three nuclear localization signals and regulates the epithelial Na+ channel (ENaC) at multiple levels ». American Journal of Physiology-Renal Physiology 299, n^o 1 (juillet 2010) : F63—F76. http://dx.doi.org/10.1152/ajprenal.00105.2010.

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We have previously reported that Dot1a is located in the cytoplasm and nucleus (Reisenauer MR, Anderson M, Huang L, Zhang Z, Zhou Q, Kone BC, Morris AP, Lesage GD, Dryer SE, Zhang W. J Biol Chem 284: 35659–35669, 2009), widely expressed in the kidney as detected by its histone H3K79 methyltransferase activity (Zhang W, Hayashizaki Y, Kone BC. Biochem J 377: 641–651, 2004), and involved in transcriptional control of the epithelial Na+ channel subunit-α gene ( αENaC) (Zhang W, Xia X, Jalal DI, Kuncewicz T, Xu W, Lesage GD, Kone BC. Am J Physiol Cell Physiol 290: C936–C946, 2006). Aldosterone releases repression of αENaC by reducing expression of Dot1a and its partner AF9 (Zhang W, Xia X, Reisenauer MR, Hemenway CS, Kone BC. J Biol Chem 281: 18059–18068, 2006) and by impairing Dot1a-AF9 interaction via Sgk1-mediated AF9 phosphorylation (Zhang W, Xia X, Reisenauer MR, Rieg T, Lang F, Kuhl D, Vallon V, Kone BC. J Clin Invest 117: 773–783, 2007). This network also appears to regulate transcription of several other aldosterone target genes. Here, we provide evidence showing that Dot1a contains at least three potential nuclear localization signals (NLSs). Deletion of these NLSs causes green fluorescent protein-fused Dot1a fusions to localize almost exclusively in the cytoplasm of 293T cells as revealed by confocal microscopy. Deletion of NLSs abolished Dot1a-mediated repression of αENaC-promoter luciferase construct in M1 cells. AF9 is widely expressed in mouse kidney. Similar to αENaC, the mRNA levels of βENaC, γENaC, and Sgk1 are also downregulated by Dot1a and AF9 overexpression. Small interference RNA-mediated knockdown of Dot1a and AF9 or aldosterone treatment leads to an opposite effect. Using single-cell fluorescence imaging or equivalent short-circuit current in IMCD3 and M1 cells, we show that observed transcriptional alterations correspond to changes in ENaC and Sgk1 protein levels as well as benzamil-sensitive Na+ transport. In brief, Dot1a and AF9 downregulate Na+ transport, most likely by regulating ENaC mRNA and subsequent protein expression and ENaC activity.

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Libbrecht, Clara, Simone Stefanie Riedel, Jessica Haladyna et Kathrin M. Bernt. « Menin Is a Therapeutic Target in MN1 High Leukemia ». Blood 132, Supplement 1 (29 novembre 2018) : 758. http://dx.doi.org/10.1182/blood-2018-99-115551.

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Abstract Meningioma-1 (MN1) has been found overexpressed in acute myeloid leukemia (AML). High MN1 expression levels are associated with poor prognosis and limited therapeutic options. Overexpression of MN1 in murine bone marrow progenitor cells causes leukemia as a single hit. Interestingly, MN1 induces a gene expression program that is reminiscent of KMT2a-rearranged leukemia, with high expression of the KMT2a (MLL-1) target genes HOXA7-13 and MEIS1. However, MN1 oncogenic functions remain unclear and no targetable therapies are available for MN1 high leukemia. We have previously shown, using a conditional knock-out mouse, that deletion of the histone methyltransferase Kmt2a increased latency and decreased penetrance of MN1 driven leukemia. We sought to determine what specific function of KMT2A is relevant to MN1 leukemogenesis in order to identify new therapeutic targets. KMT2a is a H3K4 histone methyltransferase that can perform mono, di and tri-methylation through its C-terminal catalytic site, the conserved SET domain. We first asked whether the enzymatic activity of Kmt2a was required in MN1 driven leukemia. Mice with a constitutive germline deletion of the SET domain of Kmt2a (∆SET) present some reduction of Hox gene expression during development but survive into adulthood with normal hematopoiesis. We established MN1 leukemia on a ∆SET background and found no difference in disease latency. Similarly, deletion of the SET domain did not influence colony forming potential in vitro. Menin, the product of the MEN1 suppressor gene, interacts with the N-terminal of KTM2a. The KMT2a-Menin interaction has been shown to be critical for leukemic transformation by KMT2a fusion proteins. Interestingly, this is a unique feature of leukemic cells since Menin is dispensable for steady state hematopoiesis. Since Kmt2a is required for MN1 leukemogenesis, we next investigated whether Menin was also necessary. We established MN1 leukemia on a conditional Menin knock-out background. In primary transplants, upon Cre-mediated excision of Menin, we found a slight decrease in colony forming potential as well as a modest but significant increase in survival of injected recipients. The effect of Menin deletion on MN1 leukemogenesis became much more dramatic in secondary transplants. In vitro, replating efficiency was severely impaired with increased apoptosis and cell cycle arrest. In vivo, we noted a significant increase in disease latency. Finally, we asked if Menin was a potentially relevant therapeutic target in MN1 high leukemia. In vitro, pharmacologic inhibition of the KMT2a-Menin interaction resulted in reduced growth and increased apoptosis of MN1 murine leukemia cells, at micromolar concentrations. We have identified a human leukemia cell line with high MN1 and high HOXA9 expression, Mutz3. To ensure that our findings translate to human AML, we will address the effect of Menin inhibition in Mutz3 cells as well as in patient samples with confirmed MN1 high leukemia. In summary, our data suggest that the role of Kmt2a in MN1 driven leukemia is mediated by the KMT2a-Menin interaction, identifying Menin as a druggable target in MN1 high leukemia. Disclosures Libbrecht: Institut Servier: Research Funding. Bernt:GSK: Other: husband works at GSK.

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Howard, Christina, Sergei Zari, Shuangjiang Li, Huang Huang, EunGi Kim, Se Ra Park, Trupta Purohit et al. « Abstract 3274 : Targeting NSD family histone methyltransferase activity with irreversible inhibitors ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 3274. http://dx.doi.org/10.1158/1538-7445.am2022-3274.

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Abstract Nuclear SET domain containing (NSD) family member proteins are histone methyltransferases that mono- and di-methylate histone 3 at lysine 36 (H3K36) to alter gene expression in a context-dependent manner. NSD1 and NSD3 are two members of this family which share greater than 80 percent sequence similarity in the catalytic SET domain. NSD1 is found as a fusion with NUP98 in aggressive cases of pediatric leukemia, and the lysine methyltransferase (KMTase) activity of NUP98-NSD1 drives bone marrow progenitor proliferation. Similarly, high levels of enzymatically active NSD3 mRNA are correlated with reduced disease-free survival in breast cancer and catalytic NSD3 expression is sufficient to rescue tumor growth in a xenograft model of triple negative breast cancer. Selective and potent inhibitors of NSD1 and NSD3 KMTase activity are needed to further investigate the therapeutic potential of targeting this function in leukemia and/or solid cancers. Here, we report the development of irreversible small molecule inhibitors of the NSD1 and NSD3 SET domains. We utilized a NMR-based fragment screen of the NSD1 SET domain to identify the hit BT1, which binds reversibly to the SET domain of NSD1. BT1 was extensively optimized into a series of potent irreversible ligands. Mass spectrometry and structural studies revealed that these compounds react with a cysteine near the autoinhibitory loop, providing a rational for the strong inhibition of KMTase activity we observe in biochemical assays. These inhibitors also bind to the NSD1 and NSD3 SET domains in cells and exhibit potent anti-proliferative effects against leukemia cells containing NUP98-NSD1 and breast cancer cells with high levels of NSD3 expression. In summary, we present novel irreversible inhibitors of NSD1 and NSD3 KMTase activity which represent important chemical probes that can be used to investigate the therapeutic potential of targeting NSD family member catalytic activity in cancers. Citation Format: Christina Howard, Sergei Zari, Shuangjiang Li, Huang Huang, EunGi Kim, Se Ra Park, Trupta Purohit, Hongzhi Miao, Xiaotian Zhang, Caroline Nikolaidis, Hao Li, Juliano Ndoj, HyoJe Cho, Jolanta Grembecka, Tomasz Cierpicki. Targeting NSD family histone methyltransferase activity with irreversible inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3274.

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Harrison, Christine J., Anthony V. Moorman, Claire Schwab, Nyla A. Heerema, Andrew J. Carroll et Oskar A. Haas. « Intrachromosomal Amplification of Chromosome 21(iAMP21) : Cytogenetic Characterisation and Outcome in Childhood B-Cell Precursor Acute Lymphoblastic Leukaemia (BCP-ALL). A Study On Behalf of the Ponte Di Legno International Childhood ALL Workshop ». Blood 120, n^o 21 (16 novembre 2012) : 293. http://dx.doi.org/10.1182/blood.v120.21.293.293.

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Abstract Abstract 293 Intrachromosomal amplification of chromosome 21 (iAMP21) is a distinct cytogenetic subgroup of childhood BCP-ALL, characterised by older age, low white cell count (WCC) and poor outcome on standard therapy. It is identified by a complex structure of one copy of chromosome 21, highly variable between patients, with a common 5.1Mb region of amplification within which the RUNX1 gene is located. Thus FISH, using probes directed to RUNX1 provides a reliable detection method, defining iAMP21 as ≥5 copies of RUNX1 ≥4 on the abnormal chromosome 21. The Ponte di Legno International Childhood ALL Workshop has collected data from 513 iAMP21 patients from 15 international study groups in order to further characterise this subgroup and improve identification. iAMP21 was mutually exclusive of ETV6-RUNX1 fusion (509 tested), MLL rearrangement (391 tested), TCF3-PBX1/TCF3-HLF (151 tested). There was evidence of classical high hyperdiploidy in two of the 428 patients with a successful cytogenetic result. Four patients (393 tested) were positive for the BCR-ABL1 fusion. In addition to iAMP21 all patients had one normal chromosome 21, apart from 10 patients with two copies of normal 21 in addition to iAMP21, and 4 patients with an additional iAMP21. Other whole chromosomes commonly gained or lost among the 398 patients with abnormal karyotypes were: +X (21%, n=85), −7 (5%, n=19), +10 (4%, n=17), +14 (4%, n=17), and −15 (3%, n=11). Abnormalities of 11q (14%, n=57), 7q (9%, n=35), 9p (9%, n=36) and 12p (6%, n=24) were the most frequent visible chromosomal imbalances. Deletions of CDKN2A/B and ETV6 occurred in 16% and 34% cases, respectively, suggesting that these genes were the targets of 9p and 12p abnormalities. The incidences of deletions of ETV6 (34% vs 22%) and RB1 (40% vs 6%), as well the CRLF2 rearrangement, P2RY8-CRLF2 (19% vs 4%), were significantly higher among the iAMP21 patients than a comparator cohort of 1427 children with BCP-ALL. There were a number of patients with constitutional abnormalities involving chromosome 21: Down syndrome (n=1), ring chromosome 21, r(21)c (n=3) and Robertsonian translocation between chromosomes 15 and 21, rob(15;21)(q10;q10)c (n=4). It was intriguing that in the one case with r(21)c and those with rob(15;21)(q10;q10)c, in which an acquired abnormal karyotype was present, it was the chromosome with the constitutional abnormality which gave rise to the abnormal iAMP21 chromosome. In terms of the demographic profile of iAMP21 patients, 52% were female and 48% were male, median age was 9 years (range 2–23). Patients <10 and ≥10 years old were equally distributed (51% vs 49%). The median WCC was 5×109/L, range (<1–900×109/L), with only 3% having a high WCC >50 x109/L (n=13). The NCI Risk status was standard risk (SR) in 48% and high risk (HR) in 52%. Follow-up data were available for 283 patients diagnosed before 01/01/2009. Complete remission (CR) was achieved in 280/283 (99%) of which 90 (32%) suffered a relapse and 47 (17%) patients died. In total 43% were treated as HR, with the remainder (57%) treated as SR. There was a significant improvement in event free survival (EFS) (Figure) for patients treated as HR compared to SR. After a median follow-up time of 3.4 years, the 5 year EFS rates were 70% (95% CI 59–78) and 50% (30–59), respectively, hazard ratio 0.73 (95% CI 0.59–0.91) (p=0.005). Patients with iAMP21 display a unique spectrum of genetic abnormalities, which may be useful for improving identification of iAMP21: gain of chromosomes X, 10 or 14 in the absence of high hyperdiploidy, or monosomy 7/deletion of 7q, deletions of 11q, including MLL and ATM genes, P2RY8-CRLF2, deletions of ETV6 and RB1. These characteristic abnormalities provide useful data to ascertain the presence of iAMP21 when no metaphases are available to visualise the abnormal chromosome 21, or in the rare cases where the abnormal chromosome 21 has atypical rearrangements with other chromosomes. In rare cases, iAMP21 is associated with BCR-ABL1 and high hyperdiploidy, but is exclusive of other chromosomal abnormalities of prognostic significance. Although Down syndrome was rare, other constitutional abnormalities of chromosome 21 may be related to iAMP21 in these patients. Accurate identification of this abnormality is important due to the treatment implications. This study has shown that treatment of iAMP21 patients as HR provides a significant improvement in outcome. Figure: EFS for iAMP21 patients treated as HR and SR Figure:. EFS for iAMP21 patients treated as HR and SR Disclosures: No relevant conflicts of interest to declare.

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Soverini, Simona, Angela Poerio, Alberto Ferrarini, Ilaria Iacobucci, Marco Sazzini, Joannah Score, Enrico Giacomelli et al. « Whole-Transcriptome Sequencing In Chronic Myeloid Leukemia Reveals Novel Gene Mutations That May Be Associated with Disease Pathogenesis and Progression ». Blood 116, n^o 21 (19 novembre 2010) : 885. http://dx.doi.org/10.1182/blood.v116.21.885.885.

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Abstract Abstract 885 Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) has always been regarded as a genetically homogeneous disease. However, the fact that a proportion of patients (pts), especially in the high Sokal risk setting, fail tyrosine kinase inhibitor therapy and progress to blast crisis (BC) suggests that a certain degree of heterogeneity exists. It can be hypothesized that genetic factors additional to the Ph+ chromosome may be present in these pts. To address this issue, we are currently using massively parallel sequencing to perform a qualitative and quantitative survey of the whole transcriptome of Ph+ CML cells at diagnosis and at progression to BC. Results are being integrated with genome-wide search for copy number alterations by Affymetrix SNP 6.0 arrays. We used a Solexa Illumina Genome Analyzer to scan the transcriptome of a CML patient at the time of diagnosis, at the time of remission (major molecular response) and at the time of progression from chronic phase (CP) to lymphoid blast crisis (BC). Both custom scripts and published algorithms were used for read alignment against the human reference genome, for single nucleotide variant (SNV) calling, for identification of alternative splicings and fusion transcripts, and for digital gene expression profiling. Comparison of the SNVs identified in the diagnosis and relapse samples with the SNVs detected in the remission sample – representing inherited sequence variants not specific for the Ph+ clone – allowed the identification of eight missense mutations at diagnosis affecting the coding sequences of AMPD3 (encoding adenosine monophosphate deaminase 3), SUCNR1 (succinate receptor 1), FANCD2 (Fanconi anemia, complementation group D2), INCENP (inner centromere protein), BSPRY (B-box and SPRY domain containing), HEXDC (hexosaminidase containing), NUDT9 (ADP-ribose diphosphatase) and KIAA2018 (encoding a protein with predicted DNA binding and transcriptional regulation activity) genes. Six of these mutations (FANCD2, INCENP, BSPRY, HEXDC, NUDT9) were also detected in the Ph+ clone re-emerged at the time of disease progression, together with seven additional missense mutations affecting the coding sequences of IDH2 (isocitrate dehydrogenase isoform 2), DECR1 (2,4-dienoyl CoA reductase 1), C4Orf14 (mitochondrial nitric oxide synthase), MRM1 (mitochondrial rRNA methyltransferase 1), PRKD2 (protein kinase D2), TCHP (mitostatin) and ABL1 genes. Digital gene expression analysis showed downregulation of SUCNR1, that might be a consequence of the P292A mutation we detected. IDH2, MRM1, AMPD3, and KIAA2018 mutations were found in additional pts. The IDH2 R140Q mutation was detected in 3/75 (4%) myeloid BC, 1/31 (3.2%) lymphoid BC, 0/34 Ph+ ALL and 0/23 Philadelphia-negative (Ph-) ALL pts. The MRM1 C120S mutation was found in 6/70 (9%) additional BC pts (2 lymphoid and 4 myeloid). AMPD3 and KIAA2018 genes were found to harbour the same point mutations (N334S and S1818G, respectively) in 1 out of 20 additional CP patients analyzed. Massively parallel sequencing of the sample collected at diagnosis also revealed that the Bcr-Abl kinase domain was already harbouring point mutations at low levels (E308D, A344G, R386S) but not the T315I that was selected at the time of disease progression. Point mutations in untraslated regions where miRNAs are known to bind were also detected, and are currently under validation. Digital gene expression profiling comparing progression to diagnosis showed significant expression changes including upregulation of 134 genes and downregulation of 88 genes. In particular, we observed an upregulation of the B-cell developmental factor PAX5, its interactor Lef-1 and its targets IRF4, BLNK, Bik, EBF1, CD79A, CD79B, CD19, VpreB1, VpreB3, BOB1, RAG1 and RAG2; upregulation of PAX9; upregulation of WNT3A, WNT9A, GLI3 and downregulation of SFRP1, resulting in aberrant activation of the Wnt signalling pathway. In summary, our preliminary data highlighted putative key genes whose deregulation may be recurrent in a subset of CML patients and may be linked to disease pathogenesis or progression. Their actual role in CML is currently being exlored. Massively parallel sequencing of additional patients is ongoing. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants. Disclosures: Baccarani: NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

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Renzulli, Matteo, C. Terragna, N. Testoni, E. Montanari, P. Tosi, E. Zamagni, P. Tacchetti et al. « CKS1B Over-Expression Significantly Predicts for a Lower Rate of Response to Primary Therapy with Thalidomide-Dexamethasone (Thali-Dex) for Newly Diagnosed Multiple Myeloma (MM) Patients. » Blood 108, n^o 11 (16 novembre 2006) : 371. http://dx.doi.org/10.1182/blood.v108.11.371.371.

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Abstract In MM tandem duplication and jumping translocations of the q21 band of chromosome (chr.) 1 are acquired during progression of the disease and can lead to chr.1q amplification. In several studies, chr. 1 amplification has been linked to poor prognosis after both conventional and high-dose chemotherapy. Recently, a subset of genes mapping on 1q21 band has been identified, whose increased expression may be due to increased DNA copy number. Among these genes, CKS1B has been one of the most significantly upregulated. CKS1B regulates SCF skp2 mediated ubiquitination and proteolysis of the cyclin dependent kinase inhibitor p27Kip1, whose low expression has been reported to be an independent adverse prognostic factor in MM patients. Aim of the present study was to investigate the relationship between CKS1B expression and response to primary therapy with thali-dex in a large series of patients with newly diagnosed MM. Secondary endpoint was to explore the relationship between CKS1B expression and del(13), as assessed by FISH analysis, and t(4;14), as evaluated by an RT-PCR assay designed to detect the presence of IgH/MMSET fusion gene. A total of 132 patients were analyzed. The presence of t(4;14) and CKS1B expression were investigated in all patients, while del(13) was studied in 129/132 patients. CKS1B expression was evaluated by Real-time RT-PCR. CKS1B values were separated in four different quartiles, with expression levels increasing progressively from quartile 1 to 4. Response to therapy was evaluated according to the criteria proposed by Bladè et al. The Fisher test and the Mann-Whitney test were applied for statistical analysis. On an intent-to-treat basis, the overall probability to respond (≥ partial response, PR) to up-front thali-dex therapy was 71%, while 38 patients (29%) either did not respond (NR) or progressed. Median CKS1B expression value was significantly higher in NR in comparison with patients who attained at least a PR: 1.42 (range 0.15–52.35) vs. 0.89 (range 0–11.88), respectively (p=0.01). In particular, the proportion of NR patients in the CKS1B expression quartile 4 was significantly higher as opposed to the frequency of NR in the CKS1B expression quartiles 1 to 3 (45.5% vs. 23.2%, respectively; p= 0.02). CKS1B over expression did not correlate with the presence of t(4;14) or del(13). Only 6 patients harbouring t(4;14) fell into the CKS1B expression quartile 4, as opposed to 32 patients included into the CKS1B expression quartiles 1–3 (18.2% vs. 32.3%; p, not significant). Similarly, the frequency of del(13) was comparable in the CKS1B expression quartile 4 and in the CKS1B expression quartiles 1–3 (34.4% vs. 44.3%; p, not significant). Likewise, only 2 patients carrying both t(4;14) and del(13) fell into the CKS1B expression quartile 4, as opposed to 15 patients who fell into the CKS1B expression quartiles 1–3 (6.5% vs. 15.3%; p, not significant). In conclusion, in patients with newly diagnosed MM, CKS1B over expression at baseline predicts for a significantly lower probability of response to primary remission induction therapy with thali-dex. Poor response to thali-dex conferred by CKS1B over expression is independent from the presence of t(4;14) and/or del(13). Supported by Università di Bologna, Progetti di Ricerca ex-60% (M.C.); Ministero dell’Università e Ricerca Scientifica (MIUR), progetto FIRB, RBAU012E9A_001 (M.C.); and Fondazione Carisbo.

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Iacobucci, Ilaria, Alberto Ferrarini, Marco Sazzini, Enrico Giacomelli, Annalisa Lonetti, Markus Muschen, Luciano Sumerle et al. « Whole Transcriptome Sequencing of a Philadelphia-Positive Acute Lymphoblastic Leukemia (ALL) with “Next Generation Sequencing” Technology Revealed Novel Point Mutations Associated with Disease-Progression. » Blood 114, n^o 22 (20 novembre 2009) : 3074. http://dx.doi.org/10.1182/blood.v114.22.3074.3074.

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Abstract Abstract 3074 Poster Board III-11 Background BCR-ABL1-positve Acute Lymphoblastic Leukemia (ALL) is the most common ALL subtype in adults and is associated with poor prognosis. The pathogenesis of this leukemia is related to the expression of the BCR-ABL1 fusion transcript, but additional recurrent genetic lesions are suspected to be involved in its development and progression. Aim A Next-Generation Sequencing Technology was used to sequence the whole transcriptome of leukemia cells from a BCR-ABL1-positive ALL patient at diagnosis and at relapse following tyrosine kinase inhibitor (TKI) therapy with the aim to detect acquired mutations cooperating with BCR-ABL1 in leukemia manifestation and drug-resistance. Methods Poly(A) RNA was extracted from leukemia cells and used to prepare double-stranded cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained 36 base-pair (bp) sequence reads were mapped to the reference sequence of the human genome (UCSC hg18, NCBI build 36.1) to identify single nucleotide variants (SNVs) and to estimate reads density corresponding to RNA from each known exon, canonical splice event or new candidate gene. This approach allowed us to define a detailed Digital Gene Expression (DGE) profile. Reads that showed no match to the reference genome were subsequently mapped to a dataset of all possible splice junctions created by in silico pairwise combination of the exons of all annotated genes (UCSC knownGene file) to identify alternative splicing (AS) events. Results Whole Transcriptome Shotgun Sequencing (RNA-seq) analysis generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples respectively, achieving approximately 90% diploid coverage and detecting transcripts from 62% and 64% of human annotated genes. The great majority of these active genes (78% at diagnosis and 73% at relapse) showed very low expression levels, with a number of reads per kilobase of exon model per million mapped reads (RPKM value) from 0.01 to 10, whereas 20% and 24% showed moderate expression levels (RPKM 10-100), as well as only 2% and 3% resulted highly expressed (RPKM 100-8000). Moreover, 6,390 and 4,671 AS events were also identified within 4,334 diagnosis and 3,651 relapse annotated transcripts, with the already described ALL-related Ik6 Ikaros isoform observed in both samples. Finally, 2,011 and 2,103 single nucleotide variants (SNVs) were found at diagnosis and relapse respectively, about 94% of which have been already reported in the dbSNP. Of greater interest as potential ALL-related mutations, 124 and 115 non annotated SNVs were also found at diagnosis and relapse, respectively. Of these, 43 affected both samples, while 81 and 72 resulted diagnosis and relapse private variants. In particular, the analysis was focused to the coding sequences of annotated genes, finding that non-synonymous changes were one out of the 19 shared between the two samples and affected a transmembrane receptor gene (PLXNB2). Six out of the 12 diagnosis private variants, affecting genes involved in metabolic process (PDE4DIP, EIF2S3, DPEP1, ZC3H12D, TMEM46) or transport (MVP) and 5 out of the 30 relapse private variants, affecting genes involved in cell cycle regulation (ABL1, CDC2L1), catalytic activity (CTSZ, CXorf21) or with unknown function (FAM116B). Most of these diagnosis and relapse non-synonymous private mutations resulted highly expressed, showing frequencies of mutated unique reads higher than 50%. According to this pattern, diagnosis private mutations may be carried by primary leukemic clones that did not develop again at relapse, whereas relapse private mutations have greater probability to be variants acquired during the disease progression. Interestingly, the T315I point mutation in the Abl kinase domain, that confers resistance to many TKIs, was found at relapse but not at diagnosis. Conclusions An accurate expression profile was obtained for the leukemia cells of the examined ALL patient, as well as the discovery of several new non-synonymous mutations affecting genes from different pathways and for which no correlation was previously found with ALL pathogenesis. These findings demonstrate that RNA-Seq represents a suitable and cost-efficient approach for identifying new genes potentially involved in ALL development and progression. Acknowledgments AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo 60% grants, European LeukemiaNet. Disclosures No relevant conflicts of interest to declare.

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Chu, Yajing, Yangpeng Chen, Huidong Guo, Mengke Li, Jun Shi, Tao Cheng, Feng-Chun Yang, Mingjiang Xu et Weiping Yuan. « Suv39h1 Represses the Progression of MLL-Rearranged Myeloid Leukemia Via Hoxb13 ». Blood 132, Supplement 1 (29 novembre 2018) : 3878. http://dx.doi.org/10.1182/blood-2018-99-113732.

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Abstract Acute myeloid leukemia (AML) is the most frequent and heterogeneous malignancy in adult leukemic patients. Genome-wide analyses revealed that genes involved in epigenetic modifications are among the most often re-occurring mutations in AML, suggesting a crucial role of epigenetic regulation in leukemogenesis and leukemia relapse. As a mammalian lysine methyltransferase, SUV39H1 catalyzes di- and tri-methylation of histone 3 lysine 9, and is the predominant H3K9 methyltransferase expressed in hematopoietic stem cells (HSCs). Previous studies have shown that in MLL-rearranged leukemic cells, the normal localization of Suv39h1 and Sirt1 was interrupted due to the DNA binding of Dot1L to DNA. However, the biological role of SUV39H1 in MLL-rearranged leukemia remains unexplored. In this study, we investigated the role and the underlying mechanism of Suv39h1 during leukemia progression. By analyzing the clinical databases, we found a significantly reduced expression of SUV39H1 in AML cells in comparison with normal bone marrow (BM) cells. More importantly, we found that low expression of SUV39H1 predicts poorer survival in AML patients. In MLL-fusion induced AML mouse models (MLL-AF9/MA9 and MLL-NRIP3/MN3), Suv39h1 also exhibited lower expression in leukemia stem cells (LSCs, defined as c-Kit+ or Lin-Sca1-IL-7R-c-Kit+CD34+CD16/32+ L-GMP cells) when compared with normal HSPCs. These data suggest a potential role of SUV39H1 in leukemic progression and/or maintenance. To explore if Suv39h1 functions as a tumor suppressor in MLL-fusion driven leukemogenesis, we overexpressed Suv39h1 in MA9 BM AML cells. Western blotting analysis confirmed the overexpression of Suv39h1 with a moderate increase in global H3K9me3 levels in Suv39h1-overexpressed (SUV-OE) MA9 AML cells. Interestingly, Suv39h1 overexpression prolonged the survival of recipient AML mice in both secondary and tertiary transplantation groups. Both the frequency and the absolute number of phenotypic LSCs in BM and SP were significantly reduced in SUV-OE groups as manifested by flow cytometry. Furthermore, limiting dilution assays revealed a significant six-fold decrease of functional LSCs in SUV-OE AML cells (1/314 LSCs in SUV-OE AML cells vs 1/56 in controls). Cell cycle analysis of control and SUV-OE LSCs from BM revealed a significantly decreased proportion of SUV-OE cells in the S/G2/M phase concordant by an increased proportion of G0/G1 phases when compared with control cells. In contrast, a similar apoptotic ratio of L-GMPs in BM was observed between control and SUV-OE groups. Taken together, these data demonstrated that overexpressing Suv39h1 in AML cells reduces the frequency of functional LSCs by suppression its proliferation. To explore the underlying mechanisms, gene expression profiles were assessed by RNA-Seq of SUV-OE and control mouse AML c-Kit+ cells. A total of 69 genes were differentially expressed with fold change ≥ 4. Among these genes, Hoxb13 was of particular interesting since it was reported to be recurrently mutated in several types of cancers including leukemia. ChIP-qPCR revealed a two-fold increase of H3K9m3 distribution at the promoter of Hoxb13 in SUV-OE groups, indicating Hoxb13 may be a direct downstream target of Suv39h1. Restoring the expression of Hoxb13 in SUV-OE AML cells diminished the effect of SUV-OE-mediated prolonged survival of SUV-OE AML mice. Interestingly, overexpression of Hoxb13 alone in MA9 cells had no significant effect on the survival of MA9 AML mice, indicating that Hoxb13 is a downstream effector of Suv39h1, rather than MA9, and Suv39h1 itself is a downstream mediator of MA9. To summarize, we here for the first time, demonstrate that Suv39h1 is significantly down-regulated in AMLs and could function as a tumor suppressor in MLL-rearranged leukemia by epigenetically inhibiting the Hoxb13 expression. The molecular mechanism mediated by Suv39h1-Hoxb13 axis in tumor suppression could potentially provide us novel therapeutic strategies for MLL-rearranged leukemia. YJ.C, YP.C and HD.G contributed equally to this work. Corresponding authors: WP.Y and MJ.X. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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Fiskus, Warren, Sunil Sharma, Sunil Abhyankar, Joseph McGuirk, David J. Bearss et Kapil Bhalla. « Pre-Clinical Efficacy of Combined Therapy with LSD1 Antagonist SP-2509 and Pan-Histone Deacetylase Inhibitor Against AML Blast Progenitor Cells ». Blood 120, n^o 21 (16 novembre 2012) : 868. http://dx.doi.org/10.1182/blood.v120.21.868.868.

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Abstract Abstract 868 LSD1 (KDM1A) is an FAD-dependent histone demethylase, with homology to amine oxidases. LSD1 demethylates di- and mono-methylated lysine (K) 4 on histone H3, reducing the permissive H3K4Me3 chromatin mark for gene expression. LSD1 forms a complex with the histone deacetylases (HDAC) 1 and 2 and with the co-repressor CoREST, which stimulates the activity of LSD1 toward nucleosomes. While high LSD1 expression may be an effector of blocked differentiation and confers poor prognosis in AML, LSD1 inhibition induces the expression of myeloid–differentiation associated genes and attenuates growth of AML blast progenitor cells (BPCs). Recently, LSD1 was shown to sustain the in vivo leukemogenic potential of MLL-AF9 expressing leukemia stem cells. Also, co-treatment with the LSD1 inhibitor tranylcypromine (TCP) and all-trans retinoic acid (ATRA) was shown to diminish the engraftment of primary AML BPCs in vivo in NOD-SCID-γIL-2 receptor deficient (NSG) mice. Previous studies have shown that HDAC inhibitors attenuate the levels of LSD1 through Sp1 inhibition. SP-2509 is a potent and selective FAD-binding pocket, non-MAOA and MAOB, inhibitor with an IC50 of 13 nM for LSD1. In the present studies, we determined the chromatin effects and anti-AML efficacy of SP-2509 alone and in combination with the pan-HDAC inhibitor panobinostat (PS) (Novartis Pharmaceuticals) in cultured (HL-60, OCI-AML3, MV4-11, MOLM13, THP1 and SKM1 cells) and primary human AML BPCs. Treatment with SP-2509 (250 to 1000 nM) dose-dependently increased the levels of H3K4Me2 & Me3 chromatin mark, and chromatin immunoprecipitation followed by QPCR analyses showed an increase in the H3K4Me3 mark on the gene promoters of KLF4, HMOX1, p57 and p21 in AML BPCs. SP-2509 treatment attenuated the binding of LSD1 with CoREST, accompanied with increased levels of p16, p21 and p27 in AML BPCs. Consistent with this, treatment with SP-2509 inhibited the suspension and colony growth of AML BPCs regardless of whether they expressed MLL fusion oncoproteins. Knockdown of LSD1 by shRNA also inhibited the suspension and colony growth of AML blast progenitor cells. SP-2509 also induced C/EBPα expression and features of morphologic differentiation in the cultured and primary AML BPCs. Following tail vein infusion and establishment of AML by OCI-AML3 or MOLM13 cells in NOD/SCID mice, treatment with SP-2509 (25 mg/kg b.i.w. via IP injection) for three weeks demonstrated improved survival of the mice compared to the vehicle control treated mice (p <0. 001). We have previously reported that treatment with PS depleted polycomb repressive complex proteins EZH2, SUZ12 and BMI1 but also reduced LSD1 expression in AML cells. Co-treatment with PS enhanced SP-2509-induced chromatin effects and differentiation of AML cells. Also, PS and SP-2509 synergistically induced apoptosis of the cultured AML OCI-AML3, MOLM13 and MV4-11cells (combination indices, CI <1.0). Additionally, co-treatment with SP-2509 sensitized AML cells to ATRA-induced differentiation. Notably, co-treatment with SP-2509 and PS also induced significantly greater loss of viability of primary AML BPCs but not of normal CD34+ cells. SP-2509 treatment (15 mg/kg b.i.w. IP) also dramatically improved survival of NSG mice with established human AML following tail-vein injection of primary AML blasts. Survival was further significantly improved upon co-treatment with SP-2509 and PS (5 mg/kg IP, MWF) (p < 0.001). Mice did not experience any toxicity or weight loss. Taken together, these findings demonstrate promising pre-clinical activity of combined therapy with SP-2509 and PS, warranting further in vivo development and testing of SP-2509 against human AML. Disclosures: Sharma: Salarius Pharmaceuticals: Equity Ownership.

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Cole, Christopher B., Angela M. Verdoni, David H. Spencer et Timothy J. Ley. « Dnmt3a is Required For Aberrant Self-Renewal Driven by PML-RARA ». Blood 122, n^o 21 (15 novembre 2013) : 477. http://dx.doi.org/10.1182/blood.v122.21.477.477.

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We previously identified recurrent mutations in the DNA methyltransferase DNMT3A in patients with acute myeloid leukemia (AML). DNMT3A and the highly homologous gene DNMT3B encode the two methyltransferases that are primarily responsible for mediating de novo methylation of specific CpG residues during differentiation. Loss of Dnmt3a in hematopoietic stem cells impairs their ability to differentiate into committed progenitors (Challen et al Nat Gen 44:23, 2011). Importantly, DNMT3A mutations are mutually exclusive of the favorable prognosis AML-initiating translocations, including the t(15;17) translocation (which creates the PML-RARA fusion gene), and translocations involving MLL. PML-RARA has been shown to interact with DNMT3A in vitro (Di Croce et al Science 295:1079,2002), and to require DNMT3A to induce methylation and transcriptional silencing of a subset of specific target genes. These findings, and the lack of DNMT3A mutations in APL patients, suggest that PML-RARA may require functional DNMT3A to initiate leukemia. To investigate this possibility, we utilized a well-characterized transgenic mouse model (in a pure B6 background) in which expression of PML-RARA is driven in hematopoietic stem/progenitor cells by the mouse Cathepsin G locus (Ctsg-PML-RARA+/- mice). These mice spontaneously develop acute promyelocytic leukemia (APL) with high penetrance and long latency, and also exhibit a preleukemic phenotype marked by the accumulation of myeloid cells in bone marrow and spleen. In addition, myeloid progenitor cells derived from these mice have the ability to serially replate in methylcellulose cultures, demonstrating aberrant self-renewal. We generated Ctsg-PML-RARA+/- mice lacking Dnmt3a (PML-RARA+/- x Dnmt3a-/-) as well as mice in which conditional ablation of Dnmt3b in hematopoietic cells is driven by Vav-Cre (PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+). Loss of Dnmt3a completely abrogated the ex vivo replating ability of PML-RARA bone marrow (Figure 1). Although colonies from both PML-RARA+/- and PML-RARA+/- x Dnmt3a-/- mice appeared similar in morphology and number on the first plating, PML-RARA+/- x Dnmt3a-/- marrow ceased to form colonies with subsequent replating (see Figure), and cultured cells lost the expression of the myeloid marker CD11b. The same phenotype was also observed using bone marrow from both genotypes that was secondarily transplanted into wild type recipients, indicating that it is intrinsic to transplantable hematopoietic progenitors. Reintroduction of DNMT3A into bone marrow cells derived from PML-RARA+/- x Dnmt3a-/- mice with retroviral transduction restored replating ability and CD11b expression. Competitive repopulation experiments with PML-RARA+/- x Dnmt3a-/- marrow revealed a decreased contribution to peripheral lymphoid and myeloid cells at 4 weeks, relative to PML-RARA+/- or WT control animals. Finally, 12 weeks after transplantation, recipients of PML-RARA+/- x Dnmt3a-/- bone marrow did not display an accumulation of myeloid cells in the bone marrow and spleen. Importantly, bone marrow from PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+/- mice displayed no replating deficit or loss of CD11b expression ex vivo, indicating different functions for Dnmt3a versus Dnmt3b in this model. Finally, we interrogated the effect of Dnmt3a loss on bone marrow DNA methylation patterns using a liquid phase DNA capture technique that sampled ∼1.9 million mouse CpGs at >10x coverage. Loss of Dnmt3a caused a widespread loss of DNA methylation in whole bone marrow cells, with 36,000 CpGs that were highly methylated (methylation value >0.7) in the PML-RARA+/- and WT mice, but hypomethylated (methylation value <0.4) in Dnmt3a-/- and PML-RARA+/- x Dnmt3a-/- mice. Characterization of the effect of Dnmt3a loss on leukemia latency, penetrance, and phenotype in PML-RARA+/- mice is currently being defined in a tumor watch. In summary, we have demonstrated that PML-RARA requires functional Dnmt3a (but not Dnmt3b) to drive aberrant self-renewal of myeloid progenitors ex vivo, and that loss of Dnmt3a leads to widespread DNA hypomethylation in bone marrow cells, and abrogates preleukemic changes in mice expressing PML-RARA. This data may explain why DNMT3A mutations are not found in patients with APL initiated by PML-RARA. Disclosures: No relevant conflicts of interest to declare.

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Swords, Ronan T., Aymee Perez, Ana Rodriguez, Justin M. Watts, Fernando Vargas, Roy Elias, Hugh Y. Rienhoff et Arthur Zelent. « The Small Molecule Img-98, a Potent and Selective Inhibitor of the Lysine Demethylase Lsd-1, Effectively Augments the Pro-Differentiation Effects of ATRA in a Pre-Clinical Model of AML ». Blood 126, n^o 23 (3 décembre 2015) : 460. http://dx.doi.org/10.1182/blood.v126.23.460.460.

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Abstract Acute Promyelocytic Leukemia (APL) is a cytogenetically unique subtype of acute myeloid leukemia (AML), characterized by the presence of the t(15;17)-associated PML-RARA fusion gene. This disease is curable in most patients with all-trans-retinoic acid (ATRA) based therapies, which effectively differentiate malignant promyelocytes. In patients with non-APL AML, most patients with die from their disease and ATRA has little activity. Therefore, research strategies that seek to extend the efficacy of ATRA-based treatment in AML are key avenues of investigation. From our previous studies, an epigenetic analysis of primary AML samples revealed that relative to normal CD33+ cells, loss of RARα2 expression in AML is associated with a reduction in H3K4me2 on the RARA2 promoter (a modification that is associated with transcriptional activation). The mono- and di-methyl lysine demethylase LSD1 (KDM1A) is highly expressed in patients with AML, and its overexpression has been implicated in various other tumors. Based on these data we correctly predicted that the use of small-molecule inhibitors targeting LSD1 (LSD1i) could result in epigenetic reprogramming that enhanced or facilitated the execution of the ATRA-induced differentiation program in AML cells. In the current study, we characterized a range of small molecule inhibitors of LSD-1. All the agents tested (RN-1, GSKi, SP2509, TCP, IMG-98 and OG-L002) led to inhibition of LSD-1 in a biochemical assay with varying degrees of potency. From this study, we further characterized the anti-tumor effects of IMG-98 alone and in combination with ATRA. IMG-98 is a novel LSD1 inhibitor relative to drugs of this class with comparatively different specificity, potency, pharmacokinetics, and metabolism. Its greater heavy atom count and chemical complexity contribute to these properties. By fluorine nuclear magnetic resonance (fNMR) and florescent spectrophotometry, the molecule rapidly reacts irreversibly with the FAD co-factor of LSD1 and this polypeptide is necessary to catalyze the reaction. Thermal stability shifts show the inactivated form of the enzyme becomes much more stable suggesting significant structural changes. Treatment with IMG-98 promoted the expression of the cell surface marker CD11b, associated with a differentiated immunophenotype, in both AML cell lines and primary patient material. IMG-98 produced a potent anti-proliferative effect across a range of AML cell lines and also led to growth inhibition of AML blast colony forming ability. In combination studies with ATRA, IMG-98 re-sensitized AML cells to ATRA by reactivating ATRA driven differentiation programs. Post-differentiation apoptosis was more significant for combined therapy (ATRA + IMG-98) than with either agent alone. Heatmap display of unsupervised hierarchical clustering of genes in AML cell lines differentially expressed in response to treatment with combinations of ATRA, IMG-98 or the combination, confirmed that ATRA combined with IMG-98 enhanced the expression of a subset of genes associated with the myeloid differentiation program. Updated studies on mechanisms underpinning mode of action of IMG-98 in this model will be presented. Taken together, these data demonstrate that ATRA combined with pharmacological inhibition of LSD1, may provide a promising treatment for AML by promoting differentiation and subsequent growth inhibition of AML blasts. A closely related molecule to IMG-98 is currently being optimized in late preclinical development, and clinical trials with this compound are anticipated to start in 2016. Figure 1. Comparative screening assay for LSD1 inhibition with commercially available agents (LSD1 Inhibitor Screening Assay, Cayman Chemical, Cat# 700120) Figure 1. Comparative screening assay for LSD1 inhibition with commercially available agents (LSD1 Inhibitor Screening Assay, Cayman Chemical, Cat# 700120) Disclosures Rienhoff: Imago: Employment.

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Lynch, James T., Gary J. Spencer, William J. Harris, Alba Maiques-Díaz, Filippo Ciceri, Xu Huang et Tim C. P. Somervaille. « Pharmacological Inhibitors of LSD1 Promote Differentiation of Myeloid Leukemia Cells through a Mechanism Independent of Histone Demethylation ». Blood 124, n^o 21 (6 décembre 2014) : 267. http://dx.doi.org/10.1182/blood.v124.21.267.267.

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Abstract Lysine Specific Demethylase 1 (LSD1 or KDM1A) is one of a number of epigenetic regulators which have recently emerged as candidate therapeutic targets in acute myeloid leukaemia (AML). It is a flavin adenine dinucleotide (FAD) dependent homolog of the amine oxidase family with an ability to demethylate monomethyl or dimethyl lysine 4 (K4) of histone H3, in addition to other substrates. Pharmacological inhibitors of LSD1 such as the tranylcypromine derivatives have already commenced evaluation in early phase clinical trials. While it has been widely assumed that these compounds promote differentiation of AML cells through inhibition of the demethylase activity of LSD1, the precise mechanisms by which LSD1 inhibitors function has not yet been determined. If changes in histone methylation are a central and critical mediator of the effects of LSD1 inhibitors in promoting AML cell differentiation, it would be expected that global changes in transcription would be tightly linked temporally to changes in histone methylation following drug treatment of cells. Through RNA sequencing and ChIP sequencing experiments performed in human THP1 AML cells treated for 24 hours with a potent and specific tranylcypromine-derivative LSD1 inhibitor (ORY86, trans-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropan-1-amine), we have established that wholescale up regulation of a myeloid differentiation transcription programme occurs in the absence of any significant genome-wide changes in mono- and di-methyl H3K4 and H3K9 (which are key enzymatic targets of LSD1). Thus LSD1 inhibitor-induced up regulation of myeloid differentiation gene expression is not downstream of changes in histone methylation. We further demonstrated that non-enzymatic functions of LSD1 are essential in AML cells by expressing either wild-type (WT) or catalytically inactive LSD1 (K661A) in LSD1 knockdown (KD) THP1 cells. While LSD1 KD cells exhibit myeloid differentiation and loss of clonogenic potential, both the WT and mutant versions of LSD1 were able to rescue the in vitro clonogenic potential of KD cells to an equivalent extent. Thus the histone demethylase activity of LSD1 is not required to sustain AML blasts in an undifferentiated state. Comparison of the transcriptional consequences of LSD1 inhibition with the transcriptional consequences of transcription factor knockdown in THP1 AML cells using GSEA revealed that pharmacological inhibition of LSD1 mimics depletion of GFI1. Immunoprecipitation experiments confirmed the previously described physical association of GFI1 with LSD1. Critically, the physical interactions of LSD1 with GFI1 was reversed by pharmacological inhibition of LSD1 with ORY86. Furthermore, in ChIP sequencing experiments drug treatment led to dissociation of LSD1 from promoters and enhancers. By contrast, there was no disruption of the endogenous level interaction of LSD1 with RCOR1, HDAC1 and HDAC2 (i.e. the CoREST complex) following drug treatment. To determine whether the inhibitor-induced separation of LSD1 from GFI1 is required for induction of myeloid differentiation by ORY86, we performed experiments using a GFI1-LSD1 fusion construct expressed in THP1 cells under the control of a doxycycline-regulated promoter. This construct tethers LSD1 directly to the transcription factor and circumvents any drug induced physical separation. THP1 cells expressing GFI1-LSD1 were drug resistant (as determined by immunophenotyping and clonogenic potential), in contrast to control cells expressing GFI1, LSD1 or an empty vector in the same inducible system. Thus, drug-induced physical separation of GFI1 from LSD1 is required for THP1 AML cells to undergo differentiation. Our data support a model whereby the physical association of LSD1 with transcription factors such as GFI1 is essential to maintain the differentiation block in AML. Unexpectedly, tranylcypromine-derivative inhibitors target this novel scaffolding function of LSD1, rather than its histone demethylase activity, to promote differentiation of AML cells. Disclosures Lynch: Astra Zeneca: Employment. Ciceri:Oryzon Genomics: Employment. Somervaille:Oryzon Genomics: Research Funding; Imago Biosciences: Consultancy.

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Ghelli Luserna di Rorà, Andrea, Ilaria Iacobucci, Neil Beeharry, Maria Vittoria Falzacappa, Chiara Ronchini, Cristina Papayannidis, Enrica Imbrogno et al. « Mine the Stability of the G2/M Checkpoint to Break Down Acute Lymphoblastic Leukemia Defenses Against Antineoplastic Drugs ». Blood 128, n^o 22 (2 décembre 2016) : 2808. http://dx.doi.org/10.1182/blood.v128.22.2808.2808.

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Abstract Although impressive developments have been made in the treatment of Acute Lymphoblastic Leukemia (ALL) patients, the overall survival is still very poor. With the exception of novel therapeutic strategies based on monoclonal antibodies (Bi-specific T-cell engagers, BiTEs) or immunogenic cells (CART cells), the therapeutic approaches for adult ALL patients are still base on non-selective chemotherapy or on tyrosine kinase inhibitors (TKIs) for the patients harboring the BCR-ABL1 fusion transcript. In addition a large percentage of initial successfully treated patients frequently develop relapses. Thus there is a need to improve the efficacy of conventional therapies, in particular those related to TKIs and to DNA damaging agents, in order to reduce the off-target toxicity and avoid relapses. In the present study we evaluated the in vitro, ex vivo and in vivo efficacy of MK-1775, a specific Wee1 inhibitor, in single agent and in combination with different therapeutic agents normally used for the treatment of B-/T-ALL. We firstly started by evaluated the efficacy of the compound in single agent on a panel of human B and T ALL cell lines (n=8) and on primary cells isolated from the bone marrow of adult B-ALL patients (n=8). The inhibition of Wee1 deeply reduced the cell viability and the proliferation rate, induced the apoptosis and increased the DNA damages of both leukemic cell lines and primary cells. Further cell-cycle analysis showed that in leukemic cell lines the treatment increased the number of cell in late S and G2/M phase. Light microscopy analyses, looking for nuclei morphology, confirmed that MK-1775 increased the number of mitotic cells but it interfered with normal mitotic division (induction of aberrant mitosis as showed by the increment of DNA bridges and micro-nuclei). The effects of the compound on the cell cycle profile and on the G2/M checkpoint were confirmed also in immunoblotting analyses, by the increment of phospho-HH3(ser10) and of Myt1 (mitotic isoform), and by gene expression analysis looking to specific genes involved in the G2/M checkpoints (PrimePcr DNA damage assay, Biorad). In particular genes like GADD45A and CCNB1/CCNB2 were significantly up-regulated between treated and untreated samples. Finally using a T-ALL mouse model we evaluated the effect of MK-1775 in single agent. Although no significative differences were seen between treated and un-treated samples, due to a very aggressive phenotype of the disease (all animal died after only 18 days from the engraftment), molecular analyses confirmed that the treatment induced DNA damages (increase of H2A.X and p-Chk1 ser317) and inhibited Wee1 functionality (reduction of pCDC2) on leukemic blasts isolated from both spleens and bone marrows. To evaluate if the inhibition of the G2/M checkpoint could sensitize leukemic cells to the toxicity of antineoplastic drugs, Philadelphia-negative ALL cell lines and primary leukemic cells (n=9) where treated with increasing concentration of MK-1775 and increasing concentration of the nucleotide analogue, clofarabine. Statistical analyses (Combination index value) confirmed the synergy of the combination in the reduction of the cell viability, in the inhibition of the proliferation and in the induction of the apoptosis. Similar results were seen on Philadelphia-positive ALL cell lines and primary cells (n=3) combining the MK-1775 with the TKI, bosutinib. The simultaneously inhibition of the Wee1 and the BCR-ABL downstream pathway resulted in a synergic inhibition of the cell viability, reduction of the proliferation and induction of apoptosis. In our opinion the pre-clinical results of this study are the basis for a future clinical evaluation of MK-1775 for the treatment of ALL patients. Acknowledgments: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. Disclosures Martinelli: Novartis: Speakers Bureau; BMS: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Genentech: Consultancy; Celgene: Consultancy, Speakers Bureau.

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