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The Mediator complex regulates transcription by connecting enhancers to promoters. High Mediator binding density defines super enhancers, which regulate cell-identity genes and oncogenes. Protein interactions of Mediator may explain its role in these processes but have not been identified comprehensively. Here, we purify Mediator from neural stem cells (NSCs) and identify 75 protein-protein interaction partners. We identify super enhancers in NSCs and show that Mediator-interacting chromatin modifiers colocalize with Mediator at enhancers and super enhancers. Transcription factor families with high affinity for Mediator dominate enhancers and super enhancers and can explain genome-wide Mediator localization. We identify E-box transcription factor Tcf4 as a key regulator of NSCs. Tcf4 interacts with Mediator, colocalizes with Mediator at super enhancers and regulates neurogenic transcription factor genes with super enhancers and broad H3K4me3 domains. Our data suggest that high binding-affinity for Mediator is an important organizing feature in the transcriptional network that determines NSC identity., The Mediator complex regulates transcription by connecting enhancers to promoters. Here, the authors purify Mediator from neural stem cells (NSCs), identify 75 novel protein-protein interaction partners and characterize the Mediator-interacting network that regulates transcription and establishes NSC identity.

This study examines whether active transport to the inner nuclear membrane, as shown for yeast membrane proteins Heh1 and Heh2, is conserved in metazoans. In support of this, the nuclear localization signal of metazoan Pom121 shares biochemical, structural, and functional properties with those of Heh1 and Heh2, and a Heh2-derived reporter protein targets to the inner membrane in Hek293T cells., Endoplasmic reticulum–synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2β are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2ΔNLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins.

Acute myeloid leukemia (AML) comprises a heterogeneous group of malignancies that are linked by the presence of blasts displaying morphologic and immunophenotypic features of myeloid cell differentiation. With the development of genome-wide gene expression profiling (GEP), array-base comparative genomic hybridization methodologies, and next generation sequencing technologies, the field has gained a greater understanding of the molecular features of this malignancy. Several pathologic lesions have been found to have prognostic implications contributing to a continuous refinement of risk stratification in the context of modern therapy. Recently, the Children's Oncology Group (COG)-National Cancer Institute (NCI) TARGET AML initiative molecularly characterized 993 pediatric AML cases including 197 specimens that underwent comprehensive whole genome sequencing. Of these, 94 carried one of three oncogenic fusions known to be strong drivers of leukemogenesis: RUNX1-RUNX1T1, CBFB-MYH11 and KMT2A rearrangements (KMT2Ar). Among all the alterations detected only ten occurred in more than 5% of subjects, all of which had been previously described. This suggested that low-frequency molecular subsets may exist that require larger cohorts to fully elucidate. To address this limitation, we selected 122 pediatric AML specimens that lacked RUNX1-RUNX1T1, CBFB-MYH11 and KMT2Ar by clinical testing for whole genome (WGS), exome (WES) and RNA (RNAseq) sequencing to enrich for cases that carry low-frequency events. GEP coupled with somatic mutation calls and outcome data were utilized to identify distinct molecular subtypes with prognostic implications. Structural variations, copy number alterations, single nucleotide variations and indels were determined by our established pipelines, as well as an evaluation for regulatory rearrangements driving oncogene overexpression through enhancer hijacking. In addition to known AML somatic mutations and rearrangements in genes such as CEBPA, GATA2, NPM1, WT1, FLT3, NRAS, KRAS, ETV6, Cohesin, NUP98 and KAT6A, we identified rare novel events in known oncogenic drivers. These include a GATA2-ITD as well as the repositioning of a distal MYC enhancer to ectopically activate either the MECOM or BCL11B loci. Interestingly, several AML cases carrying loss of function mutations in polycomb repressive complex 2 (PRC2) genes were found to resemble an early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) GEP by gene set enrichment analysis. ETP-ALL exhibits aberrant expression of stem cell and myeloid markers and has been shown to have a GEP consistent with transformation of a stem cell progenitor. Further, mixed phenotype acute leukemias (MPAL) with T and myeloid lineage characteristics have been previously suggested to be in this spectrum of immature leukemias. We therefore hypothesized that these PRC2-mutated AML cases represented the myeloid end of this continuum. To provide global transcriptional context to these ETP-like AMLs and evaluate a comprehensive cohort encompassing a range of pediatric myeloid malignancies, we integrated results from previously published AML, MPAL, acute megakaryoblastic Leukemia (AMKL), and ETP-ALL datasets that had RNAseq and either WES or WGS available for a total of 436 cases. t-SNE visualization using a 381 gene list derived from the top 100 most variably expressed transcripts within each cohort revealed a clear molecular classifier identifying groups that had consistent mutational compositions and disease outcomes but were agnostic of immunophenotype. This approach allowed the distinction of 63 ETP-like cases comprising a mixture of AML, MPAL, and ETP-ALL leukemias which fell into two subgroups distinguished by FLT3-ITD and PRC2 alterations. Irrespective of treatment approach, FLT3-ITD positive ETP-like leukemias enjoyed a favorable outcome whereas those with PRC2 mutations had a poor prognosis. Our data support a refined classification of pediatric myeloid malignancies based on molecular determinants that can be used for risk stratification in therapeutic trials. Disclosures Gruber: Bristol-Myers Squibb: Consultancy. Rubnitz:AbbVie: Research Funding. Reinhardt:Jazz: Other: Participation in Advisory Boards, Research Funding; CSL Behring: Research Funding; Novartis: Other: Participation in Advisory Boards; Roche: Research Funding. Locatelli:bluebird bio: Consultancy; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zwaan:Roche: Consultancy; Servier: Consultancy; Daiichi Sankyo: Consultancy; Novartis: Consultancy; Sanofi: Consultancy; Pfizer: Consultancy, Research Funding; BMS: Research Funding; Celgene: Consultancy, Research Funding; Jazz pharmaceuticals: Other: Travel support; Janssen: Consultancy; Incyte: Consultancy.

Genetic abnormalities and early treatment response are the main prognostic factors in acute myeloid leukemia (AML). Acute megakaryoblastic leukemia (AMKL) is a rare subtype of AML. Deep sequencing has identified CBFA2T3/GLIS2 and NUP98/KDM5A as recurrent aberrations, occurring in similar frequencies as RBM15/MKL1 and KMT2A-rearrangements. We studied whether these cytogenetic aberrations can be used for risk group stratification. To assess frequencies and outcome parameters of recurrent cytogenetic aberrations in AMKL, samples and clinical data of patients treated by the Associazione Italiana Ematologia Oncologia Pediatrica, Berlin-Frankfurt-Munster Study Group, Children's Oncology Group, Dutch Childhood Oncology Group, and the Saint Louis Hôpital were collected, enabling us to screen 153 newly diagnosed pediatric AMKL cases for the aforementioned aberrations and to study their clinical characteristics and outcome. CBFA2T3/GLIS2 was identified in 16% of the cases; RBM15/MKL1, in 12%; NUP98/KDM5A and KMT2A rearrangements, in 9% each; and monosomy 7, in 6%. These aberrations were mutually exclusive. RBM15/MKL1-rearranged patients were significantly younger. No significant differences in sex and white blood cell count were found. NUP98/KDM5A, CBFA2T3/GLIS2, KMT2A-rearranged lesions and monosomy 7 (NCK-7) independently predicted a poor outcome, compared with RBM15/MKL1-rearranged patients and those with AMKL not carrying these molecular lesions. NCK-7-patients (n = 61) showed a 4-year probability of overall survival of 35 ± 6% vs 70 ± 5% in the RBM15/MKL1-other groups (n = 92, P < .0001) and 4-year probability of event-free survival of 33 ± 6% vs 62 ± 5% (P = .0013), the 4-year cumulative incidence of relapse being 42 ± 7% and 19 ± 4% (P = .003), respectively. We conclude that these genetic aberrations may be used for risk group stratification of pediatric AMKL and for treatment tailoring.

T-cell acute lymphoblastic leukemia (T-ALL) is mostly characterized by specific chromosomal abnormalities, some occurring in a mutually exclusive manner that possibly delineate specific T-ALL subgroups. One subgroup, including MLL-rearranged, CALM-AF10 or inv (7)(p15q34) patients, is characterized by elevated expression of HOXA genes. Using a gene expression–based clustering analysis of 67 T-ALL cases with recurrent molecular genetic abnormalities and 25 samples lacking apparent aberrations, we identified 5 new patients with elevated HOXA levels. Using microarray-based comparative genomic hybridization (array-CGH), a cryptic and recurrent deletion, del (9)(q34.11q34.13), was exclusively identified in 3 of these 5 patients. This deletion results in a conserved SET-NUP214 fusion product, which was also identified in the T-ALL cell line LOUCY. SET-NUP214 binds in the promoter regions of specific HOXA genes, where it interacts with CRM1 and DOT1L, which may transcriptionally activate specific members of the HOXA cluster. Targeted inhibition of SET-NUP214 by siRNA abolished expression of HOXA genes, inhibited proliferation, and induced differentiation in LOUCY but not in other T-ALL lines. We conclude that SET-NUP214 may contribute to the pathogenesis of T-ALL by enforcing T-cell differentiation arrest.

Survivin plays separate roles during different phases of the cell cycle. In mitosis, Survivin is a key regulator of cell division, while in interphase, Survivin is able to protect cells from apoptosis. Survivin shuttles between nucleus and cytoplasm under the influence of one or more nuclear export signals (NESs). Paradoxically, our data show that Survivin poorly binds CRM1 in vitro because hydrophobic residues of the NES are occupied in homodimer contacts. We show that NES-preserving dimerization mutants behave as monomers in solution, show dramatically increased CRM1 binding and are more efficiently exported in vivo than wild-type Survivin. These data indicate that Survivin contains a monomer-specific NES and that dimerization modulates cytoplasmic access of the protein. Our findings have implications for both the mitotic and interphase roles of survivin.

Background One of the most important application spectrums of transcriptomic data is cancer phenotype classification. Many characteristics of transcriptomic data, such as redundant features and technical artifacts, make over-fitting commonplace. Promising classification results often fail to generalize across datasets with different sources, platforms, or preprocessing. Recently a novel differential network rank conservation (DIRAC) algorithm to characterize cancer phenotypes using transcriptomic data. DIRAC is a member of a family of algorithms that have shown useful for disease classification based on the relative expression of genes. Combining the robustness of this family’s simple decision rules with known biological relationships, this systems approach identifies interpretable, yet highly discriminate networks. While DIRAC has been briefly employed for several classification problems in the original paper, the potentials of DIRAC in cancer phenotype classification, and especially robustness against artifacts in transcriptomic data have not been fully characterized yet. Results In this study we thoroughly investigate the potentials of DIRAC by applying it to multiple datasets, and examine the variations in classification performances when datasets are (i) treated and untreated for batch effect; (ii) preprocessed with different techniques. We also propose the first DIRAC-based classifier to integrate multiple networks. We show that the DIRAC-based classifier is very robust in the examined scenarios. To our surprise, the trained DIRAC-based classifier even translated well to a dataset with different biological characteristics in the presence of substantial batch effects that, as shown here, plagued the standard expression value based classifier. In addition, the DIRAC-based classifier, because of the integrated biological information, also suggests pathways to target in specific subtypes, which may enhance the establishment of personalized therapy in diseases such as pediatric AML. In order to better comprehend the prediction power of the DIRAC-based classifier in general, we also performed classifications using publicly available datasets from breast and lung cancer. Furthermore, multiple well-known classification algorithms were utilized to create an ideal test bed for comparing the DIRAC-based classifier with the standard gene expression value based classifier. We observed that the DIRAC-based classifier greatly outperforms its rival. Conclusions Based on our experiments with multiple datasets, we propose that DIRAC is a promising solution to the lack of generalizability in classification efforts that uses transcriptomic data. We believe that superior performances presented in this study may motivate other to initiate a new aline of research to explore the untapped power of DIRAC in a broad range of cancer types. Electronic supplementary material The online version of this article (doi:10.1186/s12859-015-0737-3) contains supplementary material, which is available to authorized users.

Leucine-rich nuclear export signals (NESs) mediate rapid nuclear export of proteins via interaction with CRM1. This interaction is stimulated by RanGTP but remains of a relatively low affinity. In order to identify strong signals, we screened a 15-mer random peptide library for CRM1 binding, both in the presence and absence of RanGTP. Under each condition, strikingly similar signals were enriched, conforming to the NES consensus sequence. A derivative of an NES selected in the absence of RanGTP exhibits very high affinity for CRM1 in vitro and stably binds without the requirement of RanGTP. Localisation studies and RNA interference demonstrate inefficient CRM1-mediated export and accumulation of CRM1 complexed with the high-affinity NES at nucleoporin Nup358. These results provide in vivo evidence for a nuclear export reaction intermediate. They suggest that NESs have evolved to maintain low affinity for CRM1 to allow efficient export complex disassembly and release from Nup358.

We investigated the role of RanBP3, a nuclear member of the Ran-binding protein 1 family, in CRM1-mediated protein export in higher eukaryotes. RanBP3 interacts directly with CRM1 and also forms a trimeric complex with CRM1 and RanGTP. However, RanBP3 does not bind to CRM1 like an export substrate. Instead, it can stabilize CRM1–export substrate interaction. Nuclear RanBP3 stimulates CRM1-dependent protein export in permeabilized cells. These data indicate that RanBP3 functions by a novel mechanism as a cofactor in recognition and export of certain CRM1 substrates. In vitro, RanBP3 binding to CRM1 affects the relative affinity of CRM1 for different substrates.

Deregulated microRNA (miRNA) expression has been implicated in the pathogenesis of acute myeloid leukemia (AML). We previously showed that miR-193b is a STAT5-regulated miRNA that controls hematopoietic stem and progenitor cell (HSPC) expansion by modulating cytokine receptor signaling. Here we demonstrate that the miR-193 family members miR-193a and 193b are potent tumor suppressors in AML. Both miRNAs were downregulated in several cytogenetically-defined subgroups of pediatric and adult AML (n=202), whereas low miR-193b expression was an independent indicator for poor prognosis and survival. Accordingly, ectopic retroviral Hoxa9-Meis1 expression in HSPCs from miR-193b-/- mice resulted in a more aggressive disease with significantly shortened latency and survival as compared to miR-193bWT/WT HSPCs. Inversely, ectopic miR-193 expression in leukemic cells belonging to various AML subgroups decreased leukemic growth in vitro and prolonged survival of mice suffering from Hoxa9-Meis1-induced leukemia through a G1/S phase block. These effects were mediated by targeting c-KIT, KRAS and SOS2 - key factors of the KIT-RAS-RAF-MEK-ERK signaling cascade - as well as the downstream cell cycle regulator CCND1. Knockdown of each of these genes partially recapitulated the anti-proliferative effect of ectopic lentiviral miR-193 expression. As the tumor suppressive function is independent of patient age or AML cytogenetic background, these observations suggest an opportunistic role for miR-193 in future AML therapies. With the notion that a single miRNA can control aberrant MAPK signaling at multiple levels, restoring miR-193 expression in AML cells with constitutive activation of this cascade would assure high antileukemic efficacy, while avoiding the fast development of resistance mechanisms. Disclosures Heuser: Bayer Pharma AG: Research Funding; Novartis: Consultancy, Research Funding; BerGenBio: Research Funding; Tetralogic: Research Funding; Karyopharm Therapeutics Inc: Research Funding; Celgene: Honoraria; Pfizer: Research Funding. Mulaw:NuGEN: Honoraria. Baruchel:Jazz: Consultancy; Servier: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Baxalta: Research Funding.

CRM1 is an export receptor mediating rapid nuclear exit of proteins and RNAs to the cytoplasm. CRM1 export cargoes include proteins with a leucine-rich nuclear export signal (NES) that bind directly to CRM1 in a trimeric complex with RanGTP. Using a quantitative CRM1-NES cargo binding assay, significant differences in affinity for CRM1 among natural NESs are demonstrated, suggesting that the steady-state nucleocytoplasmic distribution of shuttling proteins could be determined by the relative strengths of their NESs. We also show that a trimeric CRM1-NES-RanGTP complex is disassembled by RanBP1 in the presence of RanGAP, even though RanBP1 itself contains a leucine-rich NES. Selection of CRM1-binding proteins from Xenopus egg extract leads to the identification of an NES-containing DEAD-box helicase, An3, that continuously shuttles between the nucleus and the cytoplasm. In addition, we identify the Xenopus homologue of the nucleoporin CAN/Nup214 as a RanGTP- and NES cargo-specific binding site for CRM1, suggesting that this nucleoporin plays a role in export complex disassembly and/or CRM1 recycling.

Background: Transport of macromolecules between the nucleus and cytoplasm of eukaryotic cells is mediated by nuclear import and export receptors. The receptors identified to date are members of a family of Ran GTPase-binding proteins whose founding member is importin- β . Interaction between these receptors and their cargo is regulated by the GTP-bound form of Ran. Export complexes form and import complexes disassemble on binding of RanGTP to the receptor. Yeast Los1p is a member of the importin- β family with a poorly defined role in tRNA production. Results: A human member of the importin- β family that is distantly related to Los1p (21% identity) has been characterized. The protein shuttled between the nucleus and cytoplasm and interacts with tRNA in a RanGTP-dependent manner. Injection of the protein into the nuclei of Xenopus oocytes resulted in a specific stimulation of the export of tRNA from the nucleus and in relief of the competitive inhibition of tRNA export caused by the introduction of saturating amounts of nuclear tRNA. Conclusions: The human protein has the functional properties expected of a transport receptor that mediates export of tRNA from the nucleus. We therefore name the protein Exportin(tRNA).

The oncogenic nucleoporin CAN/Nup214 is essential in vertebrate cells. Its depletion results in defective nuclear protein import, inhibition of messenger RNA export and cell cycle arrest. We recently found that CAN associates with proteins of 88 and 112 kDa, which we have now cloned and characterized. The 88 kDa protein is a novel nuclear pore complex (NPC) component, which we have named Nup88. Depletion of CAN from the NPC results in concomitant loss of Nup88, indicating that the localization of Nup88 to the NPC is dependent on CAN binding. The 112 kDa protein is the human homologue of yeast CRM1, a protein known to be required for maintenance of correct chromosome structure. This human CRM1 (hCRM1) localized to the NPC as well as to the nucleoplasm. Nuclear overexpression of the FG-repeat region of CAN, containing its hCRM1-interaction domain, resulted in depletion of hCRM1 from the NPC. In CAN-/- mouse embryos lacking CAN, hCRM1 remained in the nuclear envelope, suggesting that this protein can also bind to other repeat-containing nucleoporins. Lastly, hCRM1 shares a domain of significant homology with importin-beta, a cytoplasmic transport factor that interacts with nucleoporin repeat regions. We propose that hCRM1 is a soluble nuclear transport factor that interacts with the NPC.

RNA-binding proteins play a crucial role in the post-transcriptional regulation of gene expression. Polypyrimidine tract binding protein (PTB in humans) has been extensively characterized as an important splicing factor, and has additional functions in 3′ end processing and translation. ROD1 is a PTB paralog containing four RRM (RNA recognition motif) domains. Here, we discover a function of ROD1 in nonsense-mediated mRNA decay (NMD). We show that ROD1 and the core NMD factor UPF1 interact and co-regulate an extensive number of target genes. Using a reporter system, we demonstrate that ROD1, similarly to UPF1 and UPF2, is required for the destabilization of a known NMD substrate. Finally, we show through RIP-seq that ROD1 and UPF1 associate with a significant number of common transcripts.Structured summary of protein interactionsROD1 physically interacts with TMED10, RNP1, PCD6, LOC493753, RBM39, BCL7C, GST-PI, AP-3, RAE1, ASH2L2, SF3B10, SDP3, CDC23, ARL10C, CAF1B, CEP55, EXPORTIN 1, NCOR1, LENG1, SNAP29, RCBTB1, RPS10, NUP155, DYNLL2, LSF, HSPC137, TNRC6C, XPO5, TMPO, SRP19, SRI, UPF2, TRF2, SSRP1, HOXB9, C11ORF73, TAF15, WDR48, SMARCA5, CUL-2, THOC2, SEC16A, CAD, LSM2, U11/U12 snRNP, TUBA, NEZHA, TRANSPORTIN 2, CGI-135, RER1, snRP E, PRPF19, TRANSPORTIN, IER3ip1, DGCR14, RPS19BP1, TOPBP1, YIP1, CPSF6, ASF1, RBM9, ARCN1, U4/U6 snRNP, CUGbp2, WDR41, p53, DNAJC9, DAZap1, TDP43, MYL6, HEY1, RAB8B, BET1, COFILIN1, MYO12A, VAT1, PRA1, MAP7D2, MAZ, PCD7, DNAJ, GNL3L, BCAS2, NUP50, RGSip1, TRIM33B, HMG-1, RAB10, ANNEXIN A2, YKT6, TRANSFERRIN, TIM44, CTP synthase, CDC42, PPIL1, HOXA9B, GCN1, hnRNP A1, LDH-B, TRAP25, mtSSB, MED8, TIA1, HMMR, B99, H1d, IMPORTIN 5, HOX7, ZMAT5, RPR1A, MARCKS, NACA, PRMT1, HOXC9, KIAA1741, HSP90AA1, LSM14, COL1A1, PRC1, CDK2, TRX-1, CSE1, COX5B, ARL1, SEPT9, BCR/ABL, CLIP-ap1, ALY, BACH1, TRIP230, DES, GFAP, APC10, MARK2, FIP1l1, RPL38, HOXC8, GATAd2A, HSPC128, TUBB, RPL11, FAM83D, UPF1, ZFP768, RPL35A, RPL30, RAB1A, BTEbp4, RNA Pol II, SEC24B, RAN, RAC3, RPS28, RPS27, snRP A, SNF5/INI1, C1ORF35, RPS7, RPS4X, RAMA1, RPS14, TUBA6, FAM128B, T-PLASTIN, FLYWCH, MYH10, ARP1, U2AF, SERPINE1, NEFM, KIAA1826, Ki-67 and RPS11 by anti bait coimmunoprecipitation (View interaction)ROD1 physically interacts with LOC100288473, PTBP1, LOC100291593, RRP36, PCMT1, BBS9, FAM83H, ARHGEF17, VIPAR, LOC100132738, PTBP2, SACS, KBTBD5, GPR98, hnRNP F, OTUD4, CAMSAP1L1, C16ORF48, FAM64A, SCN3A, IQGAP3, ZMAT5, TEX15, NOL8, RBM4, snRNP48, TMEM33, UNC45A, OGT, DIAPH3, CDK17, LARS, FAM83D, TMTC2, HSPA1A, PCM1, CSNK1A1, PHF5A, CCDC77, PLECTIN, SETX, HSPB1, HuR, NARF, MYH2, HSP90AA1, HLTF, GSK3b, CDC42, MAGOH, TRIM21, UPF1, RBMS1, ERI1, CROCC, CSNK1D, CTNNA1, AIMP2, VPS33B, FLII, CTNNB1, eIF4G2, APC, TCF20, CYCLIN T1, HIRA, BACH1, XRCC6, HMMR, RPL9, HSD17B4, MAP2, hnRNP H1, GRIA4, FRG1, HistH1e, MSH6, CELF2, CELF1, MYBbp1A, CLASP2, STIP1, GIPC1, SF3a1, CSNK1E, CDC14A, PPIH, RBM14, DYNLL2, RALY, PUF60, TRIM33, SF3b1, TARDBP, PAPD7, SF3b2, LRRFIP2, SUPT16H, FAM110B, hnRNP Ul1, KIAA1543, AXIN1, RNP1, HERC1, BANF1, XRCC5, XPC, FYTTD1, PDCD7, TAF15, YWHAH, RB1CC1, MATR3, SRRM1, SFRS14, LAGE3, XIRP1, ARHGEF2, PRPF3, PUM1 and IQCB1 by anti bait coimmunoprecipitation (View interaction)

MicroRNAs (miRNAs) are essential for maintenance and differentiation of normal hematopoietic cells and their dysregulation is strongly implicated in leukemias. In order to identify tumor suppressor miRNAs in the context of hematological malignancies, we performed two complementary miRNA expression screenings in normal hematopoiesis as well as in childhood and adult acute myeloid leukemias (AML). We reasoned that tumor suppressor miRNAs are upregulated in mature myeloid cells, as compared to normal hematopoietic stem and progenitor cells (HSPCs), and downregulated in AML. Based on this screening strategy, we identified the miR-193 family members as potent suppressors of HSPC activity and AML growth. During normal hematopoiesis mmu-miR-193a-3p is exclusively expressed in mature myeloid cells and absent in normal HSPCs. Accordingly, in a cohort of 165 pediatric AML patients hsa-miR-193b-3p was broadly repressed throughout the cytogenetically characterized subgroups. In addition, in a cohort of 43 adult AML patients, its homolog hsa-miR-193a-3p was significantly upregulated in APL cases (p=0.0025, n=7) compared to bone marrow from healthy donors (n=5). To assess the impact of the miR-193 family members on AML maintenance and development, we lentivirally expressed miR-193a/b in the MLL-rearranged cell lines ML2 and THP1, which induced monocytic differentiation in concert with calcitriol treatment, measured by CD11b/CD14 expression (p=0.024). Consistently, enforced miR-193-expression led to a significant growth disadvantage in ML2 and THP1 cells (p= Based on these findings in AML, we further investigated the impact of the miR-193 family on normal hematopoiesis. We retrovirally expressed miR-193a in 32D cells treated with granulocyte-colony stimulating factor (G-CSF), which resulted in a strong induction of myeloid differentiation already after day 2 (p=0.006) as assessed by CD11b/Gr-1 surface marker expression. We lentivirally transduced mouse lineage negative (Lin-) HSPCs and transplanted them into irradiated isogenic recipients. Bleedings performed on weeks 4, 8 and 11, as well as the examination of the bone marrow on week 11, showed a severe competitive disadvantage of miR-193-transduced cells (week 11: 2% GFP+ miR-193- vs. 25% GFP+ miR-NSC-transduced cells). These results were further refined using highly purified ESLAM (CD45+ EPCR+ CD48− CD150+) HSCs which failed to reconstitute hematopoiesis when overexpressing miR-193a, indicated by the absence of miR-193a/GFP+ cells at week 8 post transplantation. These observations might be explained by a potent G1 cell cycle arrest in HSPCs when overexpressing miR-193a/b (4-fold decrease in the S phase population) and induction of apoptosis. Our results in normal and malignant hematopoiesis suggested that the miR-193 family acts globally through targeting relevant stem cell pathways. To validate this hypothesis we quantified the knockdown of ten predicted miR-193 target genes. qRT-PCR analysis confirmed the down regulation of KIT, KRAS, SOS2 (key components of the MAPK signaling pathway) and CCND1, a CDK regulator of G1/S phase transition. We propose a dual regulatory platform where firstly, miR-193 targets CCND1 and controls the cell cycle kinetics of stem cells. Secondly, miR-193 interferes with the KIT proto-oncogene and the RAS pathway thereby inhibiting crucial pro-proliferation and anti-apoptotic signaling cascades. Taken together, we identified the miR-193 family as a pan-tumor suppressor in childhood and adult AML. Its anti-leukemic effect is mediated by targeting the stem cell KIT/SOS2/RAS/RAF axis. Disclosures No relevant conflicts of interest to declare.

Genetic abnormalities and early treatment response are the main prognostic factors in pediatric acute myeloid leukemia (AML). Non-Down Syndrome (non-DS) acute megakaryoblastic leukemia (AMKL) is a rare subtype of AML, with a poor prognosis. These cases present with diverse cytogenetic aberrations, which might be important for risk-group stratification. A well-known genetic aberration in non-DS-AMKL is t(1;22)(p13;q13), resulting in expression of the RBM15/MKL1(OTT/MAL) fusion transcript. In recent years, targeted and genome-wide sequencing has identified novel, recurrent molecular abnormalities in pediatric AMKL. New fusions identified are inv(16)(p13.3q24.3) resulting in CBFA2T3/GLIS2 (Gruber et al, Cell, 2012; Masetti et al, Blood, 2013) and t(11;12)(p15;p13) resulting in NUP98/KDM5A (de Rooij et al, Leukemia, 2013) as specific aberrations in pediatric non-DS AMKL. Also KMT2A(=MLL)-rearrangements are recurrent in non-DS AMKL. The prognostic relevance of these novel abnormalities is not determined in a large cohort, since this disease is rare. To assess frequencies, clinical characteristics and outcome parameters of recurrent cytogenetic aberrations of pediatric non-DS AMKL, databases of the BFM-SG, DCOG, AIEOP, and COG were combined. In this study, we analyzed 151 newly diagnosed pediatric non-DS AMKL cases diagnosed between 1998 and 2014 of whom a sample was available. All patients included were screened for NUP98/KDM5A, CBFA2T3/GLIS2 and RBM15/MKL1 with reverse transcriptase(RT-) PCR,and for KMT2A-rearrangements using split signal FISH and RT-PCR. To assess outcome, probability of event-free survival (pEFS) and probability of overall survival (pOS) were estimated by the Kaplan-Meier method, and groups were compared with the log-rank test; the cumulative incidence of non-response or relapse (pCIR) was analysed by the Kalbfleisch and Prentice method, and groups were compared with the Gray's test. A Cox regression analysis was done for EFS and relapse incidence with as co-variables cytogenetic subgroup, age, sex, WBC and stem cell transplantation (time dependent variable). Patients were treated with different protocols; however, all protocols consisted of intensive chemotherapy using an anthracycline and cytarabine backbone for both induction and consolidation; 26% of patients received stem cell transplantation (SCT) in first remission. Median age was 1.6 years (range 0.1-17.1 years), with 62% of the patients younger than 2 years at diagnosis; 45% of patients were males, and median white blood cell count was 13.7x109/L (range 1.1-378.5x109/L). Translocation NUP98/KDM5A was identified in 9%; the CBFA2T3/GLIS2 translocation in 16%; RBM15/MKL1 in 12% and KMT2A-rearrangements in 9%, and hence in 54% none of these abnormalities were detected. All these aberrations were mutually exclusive. Comparing patients with NUP98/KDM5A, CBFA2T3/GLIS, RBM15/MKL1, or KMT2A-rearrangements with other pediatric AMKL patients, no significant differences in sex, age, and white blood cell count were found. Outcome between the included collaborative groups did not differ significantly. The 5-year pOS of the entire pediatric non-DS AMKL cohort was 56±4%, 5-year pEFS was 51±4%. NUP98/KDM5A, CBFA2T3/GLIS2 and KMT2A-rearranged positive patients showed a poor outcome (pOS 36±13%, 38±10%, and 38±13% respectively), compared to RBM15/MKL1 positive cases and other pediatric non-DS AMKL (70±11% and 65±5% respectively, p=0.04, Figure 1). Harboring NUP98/KDM5A, CBFA2T3/GLIS2 or a KMT2A-rearrangement resulted in an increased risk of experiencing an event (EFS; HR1.65, 95%CI;1.03-2.66, p=0.039) or relapse (RFS; HR2.14, 95%CI;1.12-4.11, p=0.022). SCT was not an independent factor for event or relapse free survival (HR1.20, 95%CI;0.65-2.20, p=0.565 and HR 1.13, 95%CI;0.57-2.24, p=0.717, respectively), nor did sex, age at diagnosis or WBC. Our results indicate that non-DS AMKL is a heterogeneous group within pediatric AML. Although the overall survival is poor for non-DS AMKL in general with a 5-yr pOS of ~55%, the poor outcome is specifically determined by cases with NUP98/KDM5A, CBFA2T3/GLIS2 and KMT2A-rearrangements. Other variants, including those with RBM15/MKL1, are associated with a better outcome. These data show that international collaboration allows the identification of prognostic subgroups, which may lead to new and more refined risk-group stratification. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

The EMBO Workshop on the Mechanisms of Nucleocytoplasmic Transport took place between 27 and 31 October 2007, in Taormina, Sicily, and was organized by E. Hurt and V. Doye. ![][1] Transport between the cell nucleus and cytoplasm has captured the imagination of researchers ever since the discovery of the cell nucleus by Robert Brown in the early 1830s (Brown, 1866). In eukaryotes, transcription and translation are spatially separated by the double membranes of the nuclear envelope. This alone necessitates a large amount of nuclear transport—that is, the export of protein‐encoding RNAs and the import of transcription factors. Besides this, many regulatory and biosynthetic pathways take place partly in the nucleus and partly in the cytoplasm, leading to a substantial flux of macromolecules between these two compartments. Most macromolecules use a nuclear import or export receptor to facilitate nuclear transport (Macara, 2001). All interphase macromolecular transport to and from the nucleus takes place through nuclear pore complexes (NPCs), which are large protein complexes composed of nucleoporins (Nups; Suntharalingam & Wente, 2003). If the nucleus is at the heart of the cell, and all transport goes through NPCs, then nuclear import and export pathways must have great importance, and the centre of the NPC might contain a crucial secret. This sense of urgency pulls together scientists from all across the world every two years to a meeting that alternates between the United States and Europe, and is unique for several reasons. First, almost the entire field is represented and the participants are mainly principal investigators (PIs). Second, everyone has the opportunity to present their work. Third, most of the data presented are unpublished (Fig 1A); these ingredients have so far resulted in a series of highly successful meetings. Figure 1. The meeting at a glance. ( A ) An important indicator to the continued success of the … [1]: /embed/graphic-1.gif

CRM1 exports proteins that carry a short leucine-rich peptide signal, the nuclear export signal (NES), from the nucleus. Regular NESs must have low affinity for CRM1 to function optimally. We previously generated artificial NESs with higher affinities for CRM1, termed supraphysiological NESs. Here we identify a supraphysiological NES in an endogenous protein, the NS2 protein of parvovirus Minute Virus of Mice (MVM). NS2 interacts with CRM1 without the requirement of RanGTP, whereas addition of RanGTP renders the complex highly stable. Mutation of a single hydrophobic residue that inactivates regular NESs lowers the affinity of the NS2 NES for CRM1 from supraphysiological to regular. Mutant MVM harboring this regular NES is compromised in viral nuclear export and productivity. In virus-infected mouse fibroblasts we observe colocalization of NS2, CRM1 and mature virions, which is dependent on the supraphysiological NS2 NES. We conclude that supraphysiological NESs exist in nature and that the supraphysiological NS2 NES has a critical role in active nuclear export of mature MVM particles before cell lysis. © 2008 by The American Society for Cell Biology.

Just as people head to the beaches for a well-deserved rest, accumulating evidence suggests that transcription factors take similar 'vacations' at the nuclear envelope. Recent studies indicate that the periphery of the nucleus provides a platform for sequestering transcription factors away from chromatin. Several transcriptional regulators, operating in different signal-transduction pathways, have been found to interact physically with components of the inner nuclear membrane. In general, this association seems to restrict access to their target genes and limit their transactivation or transrepression abilities. The mechanisms of inner nuclear membrane association are diverse, and include regulated associations with the nuclear lamina and integral membrane proteins. Together, these findings indicate that the inside of the nuclear envelope functions as a resting place for transcription factors and suggest a more direct role for the nuclear envelope in gene regulation than previously anticipated.

The nuclear pore complex (NPC) conducts macromolecular transport to and from the nucleus and provides a kinetic/hydrophobic barrier composed of phenylalanine-glycine (FG) repeats. Nuclear transport is achieved through permeation of this barrier by transport receptors. The transport receptor CRM1 facilitates export of a large variety of cargoes. Export of the preribosomal 60 S subunit follows this pathway through the adaptor protein NMD3. Using RNA interference, we depleted two FG-containing cytoplasmically oriented NPC complexes, Nup214-Nup88 and Nup358, and investigated CRM1-mediated export. A dramatic defect in NMD3-mediated export of preribosomes was found in Nup214-Nup88-depleted cells, whereas only minor export defects were evident in other CRM1 cargoes or upon depletion of Nup358. We show that the large C-terminal FG domain of Nup214 is not accessible to freely diffusing molecules from the nucleus, indicating that it does not conduct 60 S preribosomes through the NPC. Consistently, derivatives of Nup214 lacking the FG-repeat domain rescued the 60 S export defect. We show that the coiled-coil region of Nup214 is sufficient for 60 S nuclear export, coinciding with recruitment of Nup88 to the NPC. Our data indicate that Nup214 plays independent roles in NPC function by participating in the kinetic/hydrophobic barrier through its FG-rich domain and by enabling NPC gating through association with Nup88.

Pediatric acute myeloid leukemia (AML) is a heterogeneous disease and 30-40% of the patients still die. Prognosis is dependent on relevant genetic aberrations. Although many driving genetic alterations causing AML have been defined, in ~20% of the pediatric AML patients the oncogenic events remain unidentified. The ETS-Variant gene 6 (ETV6) encodes a transcription factor that functions as a tumor suppressor gene and is required for proper hematopoiesis in the bone marrow niche. Point mutations, deletions and translocations can lead to silencing of the gene, resulting in loss of transcriptional repression activity. ETV6 aberrations strongly associate with leukemia. In pediatric B-cell precursor acute lymphoblastic leukemia, translocation ETV6/RUNX1 occurs in ~25% of cases. Mutations in ETV6 are identified in ~25% of early immature T-cell ALL and also reported as event in adult AML (Van Vlierberghe et al, J Exp Med 2011; Barjesteh van Waalwijk van Doorn-Khosrovani et al, Oncogene 2005). We previously reported that pediatric AML patients can be divided in three clusters based on HOX-expression; (1) low HOXA/B expression, (2) high HOXA and low HOXB expression, and (3) high HOXA/B expression, and identified new repetitive genetic abnormalities in the third cluster, especially in NUP98. Cluster 1 is mainly represented by core-binding factor (CBF) AML, but in ~20% of these cases we did not find specific genetic abnormalities. Helton et al presented ETV6 aberrations in pediatric CBF-AML at ASH 2011, identified with whole genome sequencing, and with poor clinical outcome. We hypothesized that ETV6 aberrations might reduce the number of patients without known driving abnormality, especially in the low HOXA/B cluster. We screened a large representative de novo pediatric AML cohort for ETV6 mutations in exons 2-8 with direct sequencing, for ETV6 deletions by multiplex ligation-dependent probe amplification and for ETV6 translocations using split signal FISH, and analyzed outcome. In a well-characterized de novo pediatric AML cases with available gene-expression data, 6/275 (2.2%) patients had mutations affecting the predicted amino acid sequence of ETV6 and one had a silent mutation, 4/259 (1.5%) had an ETV6 deletion and 6/65 (9.2%) patients an MNX1/ETV6 translocation. Additionally, we identified 3 cases with a positive split signal FISH suggestive of a break in which ETV6 is involved, and a similar gene expression profile was found in these three cases. The aberrations of ETV6 were seen in patients of all three HOX-groups; n=9, n=6 and n=4 for cluster 1, 2 and 3 respectively. In patients with an ETV6 mutation (n=6) or deletion (n=4) 13 and 38 genes, respectively, were significantly up-regulated, including CLDN5,DPEP1 and BIRC7. This is consistent with the up-regulated genes in functional studies silencing ETV6 in LOUCY cells (Van Vlierberghe et al, J Exp Med 2011). High expression of BIRC7 has been associated with poor prognosis in adult acute leukemia (El-Mesallamy et al, Leuk Res 2011). The median age of patients with an ETV6-mutation or deletion (n=10) was 11.3 years (range 4.0-15.3) and 40% were female. Median WBC was significantly lower (15.1x109/L vs 47.0x109/L, p We conclude that ETV6 aberrations are rare but recurrent in pediatric AML. ETV6 aberrations predict a poor survival, although there was no evidence for an increased relapse incidence in this small cohort. Disclosures No relevant conflicts of interest to declare.

Abstract Mixed lineage leukemia (MLL) rearrangements represent about 20% of pediatric acute myeloid leukemia (AML) cases. Survival rates have increased over the past decades due to intensified chemotherapy protocols and improved supportive care. Still, approximately 30-40% will relapse during or after therapy. The role of microRNAs (miRNAs) in leukemogenesis of MLL-rearranged AML, especially towards the development of relapse is unknown. To determine whether specific miRNAs are involved in relapse development, we performed miRNA profiling, using TaqMan Low Density Array (TLDA) in pediatric AML with a special focus on MLL-rearranged cases. First, we compared miRNA profiles of de novo pediatric AML patients that relapsed with those that did not relapse. Secondly, we did the same comparison, only focussing on the MLL-rearranged subset. Third, we investigated the role of miRNAs in clonal evolution by investigating the differential expression of 6 paired initial diagnosis-relapses MLL-rearranged AML cases. An independent set of 6 paired initial diagnosis-relapse cases with MLL-rearrangements was used to confirm the identified miRNAs using single stemloop RT-qPCR. In a cohort of 127 de novo AML cases, 59/127 (46.5%) relapsed after complete remission. Comparing miRNA profiles of relapsing and non-relapsing cases, we could not identify differentially expressed miRNAs signatures between the two groups at diagnosis (p=0.643). Likewise, in a nested cohort of de novo pediatric AML MLL-rearranged cases (4 AF6, 7 AF9, 9 AF10, 1 FNBP1, 1 SEPT6, and 1 NRIP3), of which 14/23 (60.9%) relapsed we did not identify differentially expressed microRNAs (p=0.429). In the 6 paired initial diagnosis-relapse MLL-rearranged cases (2 AF9 and 4 AF10), 53 miRNAs were significantly differentially expressed (FDR Among those, miR-106b, miR-93 and miR-25, were most highly overexpressed at relapse. These microRNAs cluster together in intron 13 of minichromosome maintenance complex component 7 (MCM7) and are actively cotranscribed in the context of MCM7 primary RNA transcript. E2F transcription factor 1 (E2F1) acts as an upstream regulator. Overexpression of MCM7has been associated with poor prognosis in solid cancers and this may be linked to overexpression of the hosted microRNAs, the miR-106b~25 cluster. A possible role of the miR-106b~25 cluster in leukemia has so far not been investigated. Therefore, we validated the expression of MCM7 and E2F1 mRNA (RT-qPCR) and found them to be differentially overexpressed in the paired samples with MLL-rearrangements at relapse (n=12, p=0.006 and p=0.003, respectively). Overexpression of miR-106b and miR-25 at relapse was confirmed by stem loop RT-qPCR in the patient samples as well as in 6 additional paired samples with MLL-rearrangements at relapse (4 AF10, 1 ELL, and 1 unknown)(n=12, p Expression levels of two other predicted targets of the miR-106b~25 cluster as taken from the literature (Petrocca et al., 2008), cyclin-dependent kinase inhibitor 1A (p21WAF1/CIP1) and BCL2-like 11 (BIM), were analysed in 12 paired initial diagnosis-relapse samples. We did not find differences in mRNA expression of p21WAF1/CIP1 (p=0.17) or BIM (p=0.27) between initial diagnosis and relapse. As microRNAs regulate genes expression through translational repression or target mRNA cleavage we investigated protein level (Western blot, n=3 paired samples) of p21WAF1/CIP1, BIM, MCM7, and E2F1. In case of mRNA cleavage, a reverse correlation between predicted target gene and miRNA is expected. In 2/3 paired initial diagnosis-relapse samples the expression of p21WAF1/CIP1, BIM, E2F1, and MCM7 was downregulated in relapse samples as compared to initial diagnosis. Only one patient showed increased protein expression of E2F1 and MCM7, a modest downregulation of BIM, and no expression of p21WAF1/CIP1. Together, our data indicate that miR-106b-25 cluster may play an important role in relapse pediatric AML with MLL-rearrangements. Further research is warranted to identify the role of this cluster for clinical resistance and as treatment target. Disclosures No relevant conflicts of interest to declare.

IKAROS family zinc finger 1 (IKZF1) is a transcription factor involved in lymphoid differentiation that acts as a tumor suppressor. In B-cell precursor acute lymphoblastic leukemia (BCP-ALL) loss of IKZF1 is found in ~15% of the patients, is associated with the presence of BCR/ABL1 (t(9;22)(q34;q11)) and confers a poor clinical outcome. Recent studies suggest that IKZF1 is also involved in myeloid differentiation. The best indication that loss of IKZF1 may contribute to myeloid leukemogenesis are deletions of the short arm of chromosome 7 associated with myeloproliferative-preceded secondary acute myeloid leukemia (AML) in adults, where the commonly deleted region is mapped to the IKZF1 locus (Jager et al,Leukemia 2010). To investigate whether IKZF1 deletions play a role in pediatric AML we screened a representative well-characterized panel of 258 de novo pediatric AML samples with available gene expression data, obtained from the DCOG (The Hague, the Netherlands), the AML–Berliner-Frankfurt-Münster Study Group (Germany and Czech Republic), the Saint-Louis Hospital (Paris, France) and the Royal Hospital for Sick Children (Glasgow, UK) for deletions of the IKZF1 locus on chromosome 7p12.2 using multiplex ligation-dependent probe amplification (MLPA). Median age of the patients was 9.5 years (range 0.1-18.5 years), median white blood cell count was 46.7x109/L (range 1.2-483x109/L). All major cytogenetic subgroups were included and all patients had received intensive cytarabine-anthracycline based pediatric AML therapy. Of 11 patients with an IKZF1 deletion, 8 cases presented with monosomy 7, and 3 cases showed a focal deletion of IKZF1. These focal deletions included the complete IKZF1 gene (n=2) or exons 1-4 (n=1), leading to a loss of IKZF1 function. Focal deletions were confirmed by high-resolution array comparative genome hybridization (array-CGH). Patients with a focal deletion included a 1.5 year old boy diagnosed with AML with a fusion of MNX1/ETV6 who died from relapse; an 11.3 year old girl diagnosed with AML M5 who remains in continuous complete remission (CCR) after salvage therapy for relapse; and a 2.3 year old boy diagnosed with AML M5 in CCR. IKZF1 deleted cases (n=11) did not differ significantly from the other pediatric AML cases (n=247) with regards to age at diagnosis (median age 9.1 years compared to 9.5 years respectively, p=0.41); gender (females 55% versus 42%, p=0.41); or white blood cell count at diagnosis (median 30.2 x109/L versus 47.5 x109/L, p=0.24). No specific FAB morphology subtypes were related to IKZF1 deletions. IKZF1 deleted samples showed either none or various different additional somatic mutations, most frequently activating the RAS pathway with mutations in NRAS or PTPN11 (n=4,). In BCP-ALL IKZF1 deletions are associated with BCR/ABL1 fusions and to test if this was also true for IKZF1 deletions in AML we screened samples for the BCR/ABL1 fusion and all were negative. The 3-year pOS in IKZF1 deleted patients (n=11) was 70±14% versus 63±3% (p=0.82) in IKZF1 wild-type patients (n=231). The 3-year pEFS was 36±15% versus 46±3%, and the 3-year pCIR was 64±16% versus 36±3% (p=0.87 and p=0.09) respectively. Genes differentially expressed in monosomy 7 cases correlated significantly with gene expression changes in focal IKZF1 deleted cases when comparing significant differences to non-IKZF1-deleted cases (n=247). Genes showing increased expression in IKZF1 deleted samples included those involved in myeloid cell cycling and self-renewal, such as HEMGN, FHL2, FZD6, and SETBP1. In summary, we found focal IKZF1 deletions to be rare but recurrent events in pediatric AML. Gene expression patterns suggest that the loss of IKZF1 may be an important determinant in the biology of pediatric AML with monosomy 7. Disclosures No relevant conflicts of interest to declare.

Primary refractory and relapsed pediatric acute myeloid leukemia (AML) still lead to a significant number of childhood cancer deaths, despite the current chemotherapeutic regimens. AML leukemogenesis is driven by collaborative genetic abnormalities that induce hematopoietic maturation arrest and cell proliferation. Particular AML-associated maturation inhibiting aberrations are known to target chromatin regulators, thus directly influencing the transcriptional program of leukemic cells. Therapies targeting epigenetic processes, e.g. with hypomethylation-inducing agents, are therefore becoming an attractive therapeutic strategy in adult AML. AML biology in children is not equivalent to that of adults, thus methylation patterns seen in adult AML cannot be extrapolated to pediatric AML. Therefore there is a need to unravel the mechanism behind changes in epigenetic processes as the result of AML-causing genetic abnormalities in order to develop new drugs for pediatric AML. We hypothesized that pediatric AML samples have distinct DNA-methylation patterns which may provide a rationale for treatment with demethylating agents in specific pediatric AML subtypes. Furthermore, these differences in methylation could be characteristic for AML subgroups and that particular methylation patterns drive the expression of specific genes which may play a key role in the tumorigenesis of these AML leukemias. We performed genome-wide CpG-island methylation profiling on a representative and molecularly characterized cohort of pediatric patients with de novo AML. Empirical Bayes Wilcoxon rank-sum test showed that AML patients carrying inv(16)(p13;q22) (n=9) have distinct DNA methylation patterns when compared to non-inv(16) AML patients (n=143) (consisting mainly of MLL-rearranged, t(8;21), t(15;17), t(8;16) AML and AML cases with a normal karyotype). The MN1 gene ranked as most significantly differentially methylated in inv(16) AML compared to non-inv(16) AML, with inv(16) AML cases having significantly (p=2x10-6) lower methylation levels compared to non-inv(16) AML cases. Hypomethylation of specific regions of the MN1-associated CpG-island was confirmed by methylation specific PCR and bisulfite sequencing. Subsequent gene expression (GEP) data on 294 pediatric AML patients showed that MN1 was 8 fold higher expressed in patients carrying inv(16) compared to all other patients (9.9, n=35 vs 6.9, n=259, p Since genes known to regulate DNA methylation have frequently been shown to be mutated in adult AML we determined whether a decreased expression of DNA methyltransferases, DNMT1, DNMT2, DNMT3A, DNMT3B, could be the cause of a hypometylated MN1 locus in inv(16) AML. Our findings show that only DNMT3B expression was significantly (p=8x10-15) lower in inv(16) cases compared to non-inv(16) cases. To test whether hypomethylation of the MN1 CpG-island and the overexpression of MN1 is the result of decreased DNMT1 expression, HL60 cells which express negligible levels of MN1 were treated with the DNMT1 inhibitor Decitabine. This showed that treatment of HL60 cells with Decitabine led to increase of MN1 transcript levels, however, not as high as those observed in patient samples. This suggests that DNMT1 activity may not be the only DNA methyltransferase influencing expression of MN1 in inv(16) patients. Interestingly, we observed a high (ρs= 0.42) correlation between MN1 methylation and DNMT3B expression, which suggests DNMT3B could be an important DNA methyltransferase involved in regulating MN1expression. Overall we show that pediatric AML patients carrying and inv(16) have a characteristic DNA methylation pattern compared to other AML patients carrying specific cytogenetic aberrations. Furthermore, our data suggest that hypomethylation of the MN1 gene is an underlying mechanism for high MN1 expression in inv(16)(p13;q22) patients possibly regulated by multiple DNA methyltransferases. Disclosures No relevant conflicts of interest to declare.

Introduction In pediatric acute myeloid leukemia (AML) current survival rates are approximately 70%, but further improvements are required to improve disease outcome. Prognosis is correlated to early response to treatment and genetic aberrations (Creutzig et al, 2012). In approximately 20% no cytogenetic aberrations can be identified. In some of these cases repetitive aberrations, such as NPM1 mutations or cryptic translocations including NUP98-translocations (Hollink, 2011 and De Rooij, 2013) have been found. Recently, mutations in BCOR and BCORL1, both located on the X-chromosome, were found in adult AML using next generation sequencing. They both are transcriptional co-repressors, although with distinct binding targets (Tiacci, Heamatologica, 2012), and are thought to represent a novel mechanism of leukemogenesis. Somatic inactivating BCOR mutations were identified in 4% of adult cytogenetically normal (CN-) AML patients, predominantly located in exon 4, but also in other exons (Grossmann et al, 2011). Of interest, germline BCOR mutations cause the X-linked oculo-facio-cardio-dental genetic syndrome, which may occur due to its function as a co-repressor of the BCL6 gene. Somatic inactivating BCORL1mutations were found in 6% of adult AML patients (Li et al, 2011); all mutations were located in exon 4. Their exact role in AML and the targets of their co-repressive transcriptional activity has not been elucidated as yet (Tiacci, Haematologica, 2012). Methods We screened newly diagnosed pediatric AML patients for the presence of BCOR and BCORL1 mutations using direct sequencing of the complete coding sequence of both genes starting with a cohort of 86 patients including all cytogenetic subgroups patients, and later expanding this with an additional 146 patients for BCORL1screening of exon 4. This cohort was enriched for samples from CN-AML patients (56% and 21% respectively). Samples were obtained from the Dutch Childhood Oncology Group (DCOG; The Hague, The Netherlands), the AML-BFM-SG; Hannover, Germany and Prague, Czech Republic, and the Hôpital Robert Debré (Paris, France). Results A single BCOR mutation was found in 1 patient only with CN-AML. The mutation, p.A854T, was located in exon 4. The patient was a 4 year old boy with a FAB M1, WBC 354 x 109/L, who is alive 45 months after diagnosis. In addition, only 1 patient carried a BCORL1 mutation. The mutation, located in exon 4, p.G158X, caused a premature stop-codon. The male patient was diagnosed with secondary AML, aged 17 years, with normal cytogenetics and a WBC of 9.4 x 109/L, FAB M1, and died 3 months after diagnosis. Multiple recurrent SNPs were observed for both BCOR (rs5917933: 7/86 pts (91.9%); rs6520618: 15/86 (17.4%); rs144606152: 6/86 (7.0%)) and BCORL1 (rs4830173: 232/232 (100%); rs5932715: 36/232 (15.5%)), all in exon 4. No relation could be found between the presence of SNPs and disease outcome. Conclusions BCOR and BCORL1 mutations occur in less than 1% of pediatric AML patients. These data provide further evidence for the differences in genetic background between pediatric and adult AML. Separate next-generation studies should be performed to elucidate the genetic background of pediatric CN-AML. This project was funded by KIKA, project number 64, entitled: Aberrant signal transduction profiling in pediatric AML. Disclosures: No relevant conflicts of interest to declare.

BCR-ABL1 is frequently associated with CML and B-ALL, but is rarely found in T-ALL. We recently identified two variant ABL1 fusions in T-ALL: NUP214-ABL1, associated with episomal amplification of ABL1 in 6% of T-ALL cases, and EML1-ABL1, associated with the cryptic translocation t(9;14)(q34;q32) in 1 patient. Similar to BCR-ABL1, NUP214-ABL1 and EML1-ABL1 are constitutively activated tyrosine kinases that transform the Ba/F3 cell line to interleukin-3 (IL3) independent growth. In the case of NUP214-ABL1, however, the Ba/F3 cells need a significantly longer period to obtain the same level of proliferation compared to BCR-ABL1 and EML1-ABL1 transformed cells. In mouse bone marrow transplantation experiments, BCR-ABL1 induced CML with a latency of 3 weeks, while with EML1-ABL1 the mice developed disease after several months, and for NUP214-ABL1 no leukemia developed within 12 months after transplantation. These data suggest that EML1-ABL1 and NUP214-ABL1 are weaker oncogenes compared to BCR-ABL1. To gain further insights in these differences, we generated a number of deletion constructs of BCR-ABL1, EML1-ABL1 and NUP214-ABL1 and assayed the respective proteins for autophosphorylation and for their ability to transform Ba/F3 cells. For BCR-ABL1, we observed that the coiled-coil domain is not strictly required for kinase activity and transformation of Ba/F3 cells, as reported previously. In contrast, the coiled-coil domain of EML1 is sufficient and required to generate a constitutively activated EML1-ABL1 fusion protein. In the case of NUP214-ABL1, the coiled-coil domains are required, but not sufficient to generate an activated NUP214-ABL1 fusion in Ba/F3 cells, and also deletion of the N-terminal and C-terminal regions of NUP214 results in a loss of activity of NUP214-ABL1. Additional experiments confirmed that EML1-ABL1 is activated through homodimerization, while the exact mechanism of activation of NUP214-ABL1 remains unclear. In contrast to BCR-ABL1 and EML1-ABL1, NUP214-ABL1 seems to have a lower kinase activity and lacks detectable phosphorylation of the activation loop of the kinase domain. NUP214-ABL1 interacts with the nuclear pore proteins NUP62, NUP88 and RanBP2, is partially localized at the nuclear envelope, and phosphorylates RanBP2. In conclusion, we describe significant differences between BCR-ABL1, EML1-ABL1 and NUP214-ABL1. For BCR-ABL1, the coiled-coil domain is sufficient for kinase activation, however BCR-ABL1 can also be activated by coiled-coil independent mechanisms. These mechanisms explain the high kinase activity and strong transforming capacity of BCR-ABL1. For EML1-ABL1, the coiled-coil domain is the only domain that can activate the kinase, probably explaining its weaker transforming capacity in mouse bone marrow transplantation models when compared to BCR-ABL1. NUP214-ABL1 is a very weak oncogene, the coiled-coil domains of NUP214 need to cooperate with other domains to activate NUP214-ABL1 adequately in Ba/F3 cells. These results may explain why NUP214-ABL1 is always amplified in T-ALL patients, and why NUP214-ABL1 is associated with T-ALL and not with CML.

Background: Anti-idiotype approaches are based on the assumption that an antibody recognising a ligand can be structurally related to the receptor. Recently we have generated anti-idiotype RNA aptamers designed to mimic the human immunodeficiency virus-1 (HIV-1) Rev nuclear export signal (NES). Nuclear injection of either NES–peptide conjugates or aptamer causes the inhibition of Rev-mediated export. This implied that NES mimics and export substrate might compete for binding to the NES receptor. The mechanism of inhibition, however, is unknown. Results: The interaction between the export aptamer and CRM1 was characterised in vitro . The aptamer binds specifically to CRM1 and this interaction is sensitive to competition by Rev NES–peptide conjugates. The recognition domain of CRM1 has been mapped and includes residues found previously to affect binding of leptomycin B, a fungicide interfering with nuclear export. Conclusions: Inhibition of Rev-mediated export in vivo by export aptamers appears to result from the binding of the aptamers to the NES-recognition domain of CRM1. This observation demonstrates that anti-idiotype RNA can mimic faithfully structural and functional properties of a protein and can be used to map ligand-binding domains of receptors.

In the limited space of the nucleus, chromatin is organized in a dynamic and non-random manner. Three ways of chromatin organization are compaction, formation of loops and localization within the nucleus. To study chromatin localization it is most convenient to use the nuclear envelope as a fixed viewpoint. Peripheral chromatin has both been described as silent chromatin, interacting with the nuclear lamina, and active chromatin, interacting with nuclear pore proteins. Current data indicate that the nuclear envelope is a reader as well as a writer of chromatin state, and that its influence is not limited to the nuclear periphery.