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Limb-Girdle Muscular Dystrophy Type R1 (LGMDR1; formerly LGMD2A), characterized by progressive hip and shoulder muscle weakness, is caused by mutations in CAPN3. In zebrafish, capn3b mediates Def-dependent degradation of p53 in the liver and intestines. We show that capn3b is expressed in the muscle. To model LGMDR1 in zebrafish, we generated three deletion mutants in capn3b and a positive-control dmd mutant (Duchenne muscular dystrophy). Two partial deletion mutants showed transcript-level reduction, whereas the RNA-less mutant lacked capn3b mRNA. All capn3b homozygous mutants were developmentally-normal adult-viable animals. Mutants in dmd were homozygous-lethal. Bathing wild-type and capn3b mutants in 0.8% methylcellulose (MC) for 3 days beginning 2 days post-fertilization resulted in significantly pronounced (20–30%) birefringence-detectable muscle abnormalities in capn3b mutant embryos. Evans Blue staining for sarcolemma integrity loss was strongly positive in dmd homozygotes, negative in wild-type embryos, and negative in MC-treated capn3b mutants, suggesting membrane instability is not a primary muscle pathology determinant. Increased birefringence-detected muscle abnormalities in capn3b mutants compared to wild-type animals were observed following induced hypertonia by exposure to cholinesterase inhibitor, azinphos-methyl, reinforcing the MC results. These mutant fish represent a novel tractable model for studying the mechanisms underlying muscle repair and remodeling, and as a preclinical tool for whole-animal therapeutics and behavioral screening in LGMDR1.

Made available in DSpace on 2022-04-30T23:49:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) The recommendation of fertilization for zucchini (Cucurbita pepo) cultivation is usually made with no differentiation from the other cucurbit crops. Thus, there is a need to examine the doses of fertilizers used for zucchini production, as well as the suitability for cultivars, seasons, and cultivation regions. The aim of this study was to evaluate the influence of sulfur doses at topdressing fertilization and the supply of organic compost at planting on the production and quality of zucchini seeds. A total of eight treatments were studied in a randomized block design with a factorial scheme 4×2, corresponding to four sulfur doses at topdressing (0, 57, 114 and 173 kg ha-1 of S), in the presence (30 t ha-1) and absence of organic compost at planting. The fruits were harvested 62 DAT, and after harvest, the fruits remained in rest for 15 days in order to reach the uniformity of the physiological maturity of the seeds. The production characteristics evaluated were average fresh weight, diameter, dry weight and length of the fruits, fruit yield per plant, weight of a thousand seeds, number and seed weight. Regarding the seed quality, the percentage of germination, germination speed index, first count of the germination test, and electric conductivity, were evaluated. The supply of organic compost at planting had a positive effect on the production and physiological quality of the seeds of zucchini produced. The sulfur applied at topdressing improved the presence of the organic compost and also the production of seeds. Hence, it is recommended to apply 30 t ha-1 of organic compost at planting in association with 57 kg ha-1 of S at topdressing for higher seed yield and quality. School of Agriculture São Paulo State University School of Agriculture São Paulo State University

Shortly after the development of the CRISPR/Cas9 system, it was recognized that it is prone to induce off-target mutations at significant frequencies. Therefore, there is a strong motivation to develop Cas9 enzymes with reduced off-target activity. Multiple rational design or selection approaches have been applied to develop several Cas9 versions with reduced off-target activities (high fidelity). To make these high-fidelity Cas9s available for model systems other than human cells and bacterial strains, as, for example, in zebrafish, new specialized expression vectors need to be developed. In this chapter, we focused on the HypaCas9 and HiFi Cas9 high-fidelity enzymes and incorporated the mutations of these Cas9 versions into a codon-optimized zebrafish Cas9 vector. This optimized vector was further improved by introducing an artificial polyadenine insert (A71) since polyadenylation is known to enhance mRNA translational efficiency. The Hypa-nCas9n and HiFi-nCas9n vectors were produced by single-site mutagenesis from pT3TS-nCas9n-A71 vector. We then tested the polyadenylated mRNAs for nCas9n, Hypa-nCas9n, HiFi-nCas9n, and HiFi-Cas9 protein for editing efficiency in five genome editing strategies and found that these high-fidelity Cas9 versions had different performances ranging from activity at 2-4 sites, where the wild-type nCas9n is active, indicating that these Cas9 versions have different sgRNA preferences. In summary, the developed new high-fidelity Cas9 vectors will enable researchers to perform much more accurate genome editing.

Shortly after the development of the CRISPR/Cas9 system, it was recognized that it is prone to induce off-target mutations at significant frequencies. Therefore, there is a strong motivation to develop Cas9 enzymes with reduced off-target activity. Multiple rational design or selection approaches have been applied to develop several Cas9 versions with reduced off-target activities (high fidelity). To make these high-fidelity Cas9s available for model systems other than human cells and bacterial strains, as, for example, in zebrafish, new specialized expression vectors need to be developed. In this chapter, we focused on the HypaCas9 and HiFi Cas9 high-fidelity enzymes and incorporated the mutations of these Cas9 versions into a codon-optimized zebrafish Cas9 vector. This optimized vector was further improved by introducing an artificial polyadenine insert (A71) since polyadenylation is known to enhance mRNA translational efficiency. The Hypa-nCas9n and HiFi-nCas9n vectors were produced by single-site mutagenesis from pT3TS-nCas9n-A71 vector. We then tested the polyadenylated mRNAs for nCas9n, Hypa-nCas9n, HiFi-nCas9n, and HiFi-Cas9 protein for editing efficiency in five genome editing strategies and found that these high-fidelity Cas9 versions had different performances ranging from activity at 2-4 sites, where the wild-type nCas9n is active, indicating that these Cas9 versions have different sgRNA preferences. In summary, the developed new high-fidelity Cas9 vectors will enable researchers to perform much more accurate genome editing.

Inherited bone marrow failure syndromes (IBMFS) are a group of rare inherited genetic disorders defined by impaired hematopoiesis and multi- or unilineage cytopenias. Dyskeratosis Congenita (DC) is characterized by pancytopenia, abnormal skin pigmentation and leukoplakia and is caused by mutations in genes involved in telomere biogenesis and/or RNA processing such as TERT, TERC, DKC1 and PARN. Shwachman-Diamond syndrome (SDS) is characterized by neutropenia and exocrine pancreatic insufficiency and occurs as a result of mutations in genes required for ribosome subunit maturation such as SBDS, DNAJC21 and EFL1. How the disruption of such ubiquitous cellular processes leads to distinct cytopenic phenotypes is not fully understood. Further, IBMFS patients have a high risk of developing myelodysplastic syndrome and/or acute myeloid leukemia with a cumulative incidence of 36% by 30 years of age for SDS patients and 13% for DC patients who do not undergo a stem cell transplant. As IBMFS are rare and large numbers of primary human samples are not readily available for mechanistic studies, we employed zebrafish (Danio rerio) given their highly conserved hematopoietic program and ease of genetic manipulation. Using CRISPR-Cas9 genomic editing, we generated deletion mutations predicted to introduce premature stop codons in the zebrafish orthologs, parn and dnajc21, two more recently identified causes of DC and SDS, respectively. Homozygous zygotic parn mutants had normal morphology and were viable to adulthood. However, fertile homozygous adult females were not recoverable in subsequent generations, implicating parn as an essential factor in oocyte specification. In contrast, homozygous dnajc21 mutants showed normal development to adulthood. Using whole mount in situ hybridization (WISH), we found increased expression of hematopoietic precursor markers (erythroid - gata1 and myeloid - lcp1) and concurrently decreased expression of mature hematopoietic markers (erythroid - hbbe3, myeloid - mpx and thrombocyte - CD41) in parn mutants at 24- and 48-hours post-fertilization (hpf). Further, o-dianisidine staining showed reduced hemoglobinized erythrocytes at 48 hpf. These data indicate that multilineage embryonic hematopoiesis is compromised in parn mutants, recapitulating the pancytopenia observed in patients with DC. WISH for mpx in dnajc21 mutants revealed reduced expression at 24 and 48 hpf, recapitulating the neutropenia seen in SDS. Activation of the TP53 tumor suppressor pathway has been suggested to mediate marrow failure and leukemic progression in some types of IBMFS. Using quantitative PCR to measure the expression of tp53 and its downstream effector p21 at 48 hpf, we observed no significant changes in parn mutants but significantly upregulated tp53 expression in dnajc21 mutants. To further study the role of tp53 in dnajc21-mutant SDS, we crossed dnajc21 mutants with a zebrafish line carrying a tp53 R217H point mutation that confers anti-apoptotic phenotypes. Heterozygous tp53 loss in a dnajc21-/- or +/- background resulted in reduced overall larval growth and abnormal yolk sac development, suggesting defective lipid metabolism. Using the lipophilic dye, Oil Red O, we observed reduced lipid distribution in the vasculature and caudal hematopoietic tissue region (equivalent to mammalian fetal liver) of dnajc21 mutants at 48 hpf. In summary, these findings support a role for PARN in hematopoietic lineage specification through mechanisms that are predominantly TP53-independent. By contrast, DNAJC21 is required for neutrophil specification and normal lipid metabolism and may function in a TP53-dependent manner. These zebrafish models provide new insights into the unique biology underlying these IBMFS and can serve as an in vivo platform for identifying therapeutic compounds that restore normal hematopoiesis and prevent leukemic transformation. Disclosures Dror: Alexion Canada: Other: Received funding for a Marrow Failure and Myelodysplasia conference that I organized April 2021; RepaetDiagnostic Laboratory: Other: Received funding for a Marrow Failure and Myelodysplasia conference that I organized April 2021. Berman: Oxford Immune Algorithmics: Membership on an entity's Board of Directors or advisory committees.

The aquatic macrophytes removed from hydroelectric power plants, as well as boiler ash derived from burning sugarcane bagasse can provide nutrients and recover degraded soils more quickly. Thus, this study aimed to evaluate the chemical attributes of a degraded soil in recovery process with mechanical preparation, with or without addition of organic residue (aquatic macrophytes) or agroindustrial residue (ash from sugarcane bagasse), compared to a degraded area without interference in the time of soil removal and in a conserved Cerrado area. The experimental design was a randomized block with four replications. The treatments were: conserved Cerrado soil (T1), area of degraded soil without human intervention since their degradation (T2), soil with mechanized preparation without addition of residue (T3), soil with mechanized preparation and addition of 32 Mg ha-1 of organic residue (T4), soil with mechanical preparation and addition of 45 Mg ha-1 of agroindustrial residue (T5). The orthogonal contrasts analyzed were C1 [(T2+T3+T4+T5)/4 -T1], C2 [(T2+T3) - (T4+T5)], C3 [(T2) -(T3)] and C4 [(T4) - (T5)]. In general, the chemical attributes of degraded soil increased after the incorporation of organic and agroindustrial residue, which is higher than the soil of the conserved Cerrado area. The boiler ash contributed most, to increase the nutrient content and fertility of the degraded soil. Key words: Aquatic macrophytes, ash from sugarcane bagasse, degraded soil, fertility, soil tillage.

Long intergenic noncoding RNA-p21 (lincRNA-p21) is a long noncoding RNA transcriptionally activated by tumor protein p53 (TP53) and hypoxia inducible factor 1 alpha subunit (HIF1A). It is involved in the regulation of TP53-dependent apoptosis and the Warburg effect. We have investigated the role of lincRNA-p21 in NSCLC.LincRNA-p21 expression was assessed in tumor and normal tissue from 128 patients with NSCLC and correlated with time to relapse and cancer-specific survival (CSS). H23, H1299, and HCC-44 cell lines were cultured in hypoxic conditions after silencing of lincRNA-p21. The TaqMan human angiogenesis array was used to explore angiogenesis-related gene expression. Levels of the protein vascular endothelial growth factor A were measured by enzyme-linked immunosorbent assay in the cell supernatants. Angiogenic capability was measured by human umbilical vein endothelial cell tube formation assay. Microvascular density in tumor samples was analyzed by immunohistochemistry.LincRNA-p21 was down-regulated in tumor tissue, but no association was observed with TP53 mutational status. High lincRNA-p21 levels were associated with poor CSS in all patients (p = 0.032). When patients were classified according to histological subtypes, the impact of lincRNA-p21 was confined to patients with adenocarcinoma in both time to relapse (p = 0.006) and CSS (p0.001). To explain the poor outcome of patients with high lincRNA-p21 expression, we studied the role of lincRNA-p21 in angiogenesis in vitro and observed a global downregulation in the expression of angiogenesis-related genes when lincRNA-p21 was inhibited. Moreover, supernatants from lincRNA-p21-inhibited cells were significantly less angiogenic and had lower levels of secreted vascular endothelial growth factor A than controls did. Finally, tumor samples with high lincRNA-p21 levels had higher microvascular density.Our findings suggest that lincRNA-p21 affects outcome in patients with NSCLC adenocarcinoma through the regulation of angiogenesis.

Epigenetic mechanisms are crucial for the inactivation of key genes related to the survival of Hodgkin lymphoma (HL) cells, and methylation is a frequent epigenetic mechanism of microRNA silencing. We have examined the methylation-induced silencing of tumor suppressor microRNAs in HL cell lines and confirmed our results in patient lymph nodes. In addition, we evaluated the in vitro effectiveness of 5-aza-2-deoxycytidine (5-Aza-dC) in HL cell lines. Ten microRNAs containing CpG islands in their promoter region were re-expressed in both the L-428 and L-1236 cell lines. Interestingly, miR-34a and miR-203, both known tumor suppressor microRNAs, were found to be methylated in cell lines and in patient samples. 5-Aza-dC treatment resulted in a dose-dependent antiproliferative effect at 72 h in all the HL cell lines. In summary, 5-Aza-dC treatment of HL cell lines inhibits proliferation at high doses and produces re-expression of the tumor suppressor microRNAs at low-intermediate doses.

Background and Objectives Surgery is the standard treatment for colorectal cancer (CRC), and adjuvant chemotherapy has been shown to be effective in stage III but less so in stage II. We have analyzed the expression of the miR-200 family in tissue samples from resected CRC patients and correlated our findings with survival to adjuvant treatment with fluoropyrimidines. Methods Tumor tissue samples were obtained from 127 surgically resected patients with stage I–III CRC. miRNA detection was performed using TaqMan MicroRNA assays. Results High levels of miR-200a and miR-200c were associated with longer overall survival, while high levels of miR-429 correlated with longer overall and disease-free survival (DFS). In the subgroup of 56 patients treated with fluoropyrimidines and in the smaller subgroup of 32 stage II patients treated with fluoropyrimidines, those with high levels of miR-200a, miR-200c, miR-141, or miR-429 had significantly longer overall and DFS. Low miR-429 levels were identified as an independent prognostic marker. High levels of miR-429 combined with 5-fluorouracil inhibited cell invasion in LOVO cells. Conclusions miR-200a, miR-200c, miR-141, and miR-429 expression levels may identify CRC patients, including those with stage II disease, who are most likely to benefit from adjuvant chemotherapy. J. Surg. Oncol 2014; 109:676–683. © 2014 Wiley Periodicals, Inc.

Inherited bone marrow failure syndromes (IBMFs) are a group of rare genetic disorders characterized by deficient hematopoiesis and extra-hematologic traits. Most known entities are related to a specific gene or group of genes, but others still remain unclassified. In many cases, the involved proteins are required for critical processes involved in cell survival, such as ribosome biogenesis, maintenance of telomere length, and DNA repair. Importantly, patients with IBMFs have higher risks of developing a variety of cancers from leukemia to solid tumours of the head and neck. In 2015, we published four patients from three different families with mutations in the Poly(A)-specific ribonuclease (PARN) gene. This gene encodes a ribonuclease which is involved in degradation of the poly(A) tails, which regulate mRNA turnover, and thus gene expression. Three of the patients presented with several degrees of mental illness and/or developmental delay. The fourth patient, harbored both a monoallelic deletion and a point mutation at the catalytic domain of the protein, and presented with bone marrow failure and hypomyelination, similar to a severe form of dyskeratosis congenital (DC) known as Hoyeraal-Hreidarsson syndrome. In the last two decades, zebrafish (Danio rerio) has emerged as an excellent animal model for human disease, and is especially relevant in hematology, since many of the transcription factors and cell types are highly conserved. Zebrafish have a single PARN ortholog,with 64% sequence identity to the human gene. Using CRISPR-Cas9 genome editing and a combination of six sgRNAs, we generated a 1.2 kb deletion in the zebrafish parn ortholog extending from exon 5 to 13, causing a premature stop codon. Homozygous fish were generated by incrossing to replicate the complete loss-of-function observed in the patient with the DC-like phenotype. Using whole-mount in situ hybridization (WISH) at 48 hours post-fertilization (hpf), we observed a decrease in the number of several mature myeloid cell lineages including neutrophils (labeled with mpx; p PARN is described as a protein involved in RNA processing, but has also been associated with telomere maintenance. This latter process is crucial for cell senescence and genome integrity, and is a known cause of several IBMFSs. The telomerase ribonucleoprotein complex is highly active in hematopoietic stem and progenitor cells (HSPCs) and plays a role in cell differentiation. This complex is composed of a reverse transcriptase (TERT), RNA template (TERC), and the dyskerin protein complex (DKC1), mutations of which represent a common cause of DC. qPCR analysis in zebrafish parn mutants revealed a 2.98-fold reduction in tert expression compared with the wild type fish. Combined, these findings suggest that PARN plays an important role in HSC differentiation into myeloid and erythroid lineages, resulting in a bone marrow failure phenotype. Our model provides a unique in vivo platform to study the role of PARN in hematopoiesis and for identifying compounds that restore normal blood cell ratios, which may have the potential to prevent future leukemic transformation. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a heterogeneous disease whose prognosis is mainly related to the biological risk conferred by cytogenetics and molecular profiling. In elderly patients (>= 60 years) with normal karyotype AML miR-3151 have been identified as a prognostic factor. However, miR-3151 prognostic value has not been examined in younger AML patients. In the present work, we have studied miR-3151 alone and in combination with BAALC, its host gene, in a cohort of 181 younger intermediate-risk AML (IR-AML) patients. Patients with higher expression of miR-3151 had shorter overall survival (P = 0.0025), shorter leukemia-free survival (P = 0.026) and higher cumulative incidence of relapse (P = 0.082). Moreover, in the multivariate analysis miR-3151 emerged as independent prognostic marker in both the overall series and within the unfavorable molecular prognostic category. Interestingly, the combined determination of both miR-3151 and BAALC improved this prognostic stratification, with patients with low levels of both parameters showing a better outcome compared with those patients harboring increased levels of one or both markers (P = 0.003). In addition, we studied the microRNA expression profile associated with miR-3151 identifying a six-microRNA signature. In conclusion, the analysis of miR-3151 and BAALC expression may well contribute to an improved prognostic stratification of younger patients with IR-AML., Marina Diaz-Beya is supported by ISCIII (Rio Hortega CM13/00205). This research was in part supported by Fundacion Espanola de Hematologia y Hemoterapia (beca de investigacion MDB). This research is also supported by grants from Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III PI13/00999 (PI: JE), RETICS RD12/0036/0010 (JE; MDB) and SDCSD from School of Medicine, University of Barcelona, AECC-Catalunya 2013 (AN) (sponsored by Mat Holding), and grants AGAUR 2014SGR-1281, ISCIII RD12/0036/0071 and PI014/00450 (JS). Anna Cordeiro is an APIF fellow of the University of Barcelona.

NKX2-1, a key molecule in lung development, is strongly expressed in lung cancer, especially in adenocarcinoma patients. Recently, two microRNAs interacting with NKX2-1 have been described: miR-365 that down-regulate NKX2-1; and miR-33a that is directly upregulated. To date, in non-small cell lung cancer (NSCLC) contradictory prognostic information about NKX2-1 and its microRNAs has been described. Aim: Elucidate the prognostic role of NKX2-1 and its microRNAs in resected NSCLC patients. Methods: mRNA and microRNA expression was determined using TaqMan assay in 142 NSCLC patients. The cohort was molecularly characterized including mutational status of TP53, EGFR and KRAS. Results: Locally advanced stages (III) had higher NKX2-1 expression than early stages (I-II) (p=0.002). High NKX2-1 expression was associated with longer disease-free survival (DFS) only in early stages (p=0.0389). Regarding to TP53 status, NKX2-1 was significantly downregulated in TP53-mutated patients (p=0.0087). Interestingly, TP53-WT patients with high expression of NKX2-1 had longer overall survival (OS) (p=0.0274) and longer DFS (p=0.00276). A negative correlation between NKX2-1 and miR-365 was found (r=-0.342, p Conclusions: In our cohort NKX2-1 impacts prognostic in early stage NSCLC particularly in patients without TP53 mutation, but no prognostic value was confirmed for its associated microRNAs. Supported by PFR Hospital Clinic de Barcelona, Becario SEPAR 2015, Mutual Medica 2015.

Introduction: JAK/STAT signaling is of major importance for hematopoiesis and immune function. A heterozygous JAK1 c.1901C>A de novo point mutation was identified in three members of a family (a mother and two male sons). These patients presented with a unique clinical phenotype of severe atopic dermatitis, markedly elevated peripheral blood eosinophil counts with eosinophilic infiltration of the liver and gastrointestinal tract, hepatosplenomegaly, and failure to thrive. Treatment with ruxolitinib, a JAK1/2 inhibitor, resulted in reduced eosinophilia and improved growth (Del Bel et al. JACI 2017). However, the role of this JAK1 mutation in hematopoiesis remains unclear. Methods: ubi-JAK1-WT or A634D-P2A-sfGFP-pA zebrafish transgenic lines were generated incorporating the human genes. Both of these constructs were injected into embryos, but the survival rate for the JAK1-A634D embryos was very low. Six hour post-fertilization (hpf) embryos expressing the mutant transgene were treated with 1 µM ruxolitinib for 24h, which dramatically increased survival. Whole-mount in situ hybridization (WISH) was used to assess changes in hematopoietic stem cells/blood cell populations due to wild-type or JAK1-A634D expression at 24, 28, 36 and 48 hpf using a battery of blood lineage specific probes. O-dianisidine staining was employed to evaluate hemoglobin levels. ImageJ cell counter pluggin was used for cell quantification. Gata1 and o-dianisidine staining intensity was analyzed using Ilastik and CellProfiler. Six groups of 30-50 embryos were analyzed by RNA sequencing (RNAseq): control, JAK1-WT and JAK1-A634D at 28 and 36 hpf. RNA was extracted using Trizol. RNAseq data was analyzed with edgeR and enrichment analysis was performed using DAVID 6.8. Results: JAK1-A634D was toxic to zebrafish embryos but they survived to sexual maturity post-ruxolitinib therapy. F1 embryos from JAK1-A634D founders with high expression of the transgene exhibited abnormal development (70% of embryos) which could be reduced to 12% of embryos following 24h exposure to 1 µM ruxolitinib. This rescue suggests that JAK1-A634D is highly active in zebrafish and its activity needs to be under tight regulation for normal development. Both JAK1-WT and A634D mutant fish demonstrated increased HSPCs (c-myb/runx1 expression) at 36 hpf (pWT>controls; p=0.0308 and p=0.0045, respectively). pu.1/spi1 early myeloid expression showed a similar expression pattern at 28 hpf, A634D>WT>controls; p Conclusions: JAK1-A634D plays an important role in zebrafish development and specifically impacts the myeloid lineage, particularly the granulocyte branch, from which eosinophils arise. Ruxolitinib can revert the arrested development phenotype, demonstrating the specificity of this targeted therapy. Our model provides the first in vivo evidence of the effects of the JAK1-A634D mutation on hematopoiesis and will provide further insight in understanding the cause of familial hypereosinophilia. Disclosures No relevant conflicts of interest to declare.

Background The potential benefit of adjuvant chemotherapy in surgically resected patients with stage II colorectal cancer is controversial. The current guidelines, which are based solely on clinical factors, have limited usefulness, and a clear need exists for biomarkers to supplement the clinical information. MicroRNAs (miRNAs) have previously been shown to be useful cancer biomarkers. In the present study, we assessed the usefulness of a miRNA score to help identify the subset of high-risk patients likely to benefit from adjuvant chemotherapy. Patients and Methods Six miRNAs previously identified as prognostic markers in Asian patients (miR-21-5p, miR-20a-5p, miR-103a-3p, miR-106b-5p, miR-143-5p, and miR-215) were studied in tumor samples from 71 white patients with stage II colon cancer. Results Three miRNAs (miR-103a-3p, miR-143-5p, and miR-215) emerged as independent prognostic markers on multivariate analysis and were used to construct a miRNA-based score that classified patients into high- and low-risk groups. The patients in the high-risk group had significantly shorter disease-free survival compared with their low-risk counterparts (P = .003). The time-dependent receiver operating characteristic curve analysis showed that our 3-miRNA score improved the prediction of outcome when added to the clinical features (P = .023). Conclusion Our 3-miRNA score added valuable prognostic information to the clinical features in stage II colon cancer. Further research in this field could provide useful tools to determine whether adjuvant chemotherapy would benefit patients with stage II colon cancer after surgery.

// Alfons Navarro 1 , Rut Tejero 1 , Nuria Vinolas 2 , Anna Cordeiro 1 , Ramon M. Marrades 3 , Dolors Fuster 1 , Oriol Caritg 1 , Jorge Moises 3 , Carmen Munoz 1 , Laureano Molins 5 , Josep Ramirez 4 , Mariano Monzo 1 1 Molecular Oncology and Embryology Laboratory, Human Anatomy Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain 2 Department of Medical Oncology, Institut Clinic Malalties Hemato-Oncologiques (ICMHO), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, Barcelona, Spain 3 Department of Pneumology, Institut Clinic del Torax (ICT), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, CIBER de Enfermedades Respiratorias (CIBERES), Barcelona, Spain 4 Department of Pathology, Centro de Diagnostico Biomedico (CDB), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, CIBERES, Barcelona, Spain 5 Department of Thoracic Surgery, Institut Clinic del Torax (ICT), Hospital Clinic de Barcelona, University of Barcelona, Barcelona, Spain Correspondence to: Alfons Navarro, e-mail: anavarroponz@ub.edu Keywords: PIWI proteins, piwiRNAs, PIWIL1, PIWIL4, NSCLC Received: November 14, 2014 Accepted: December 21, 2014 Published: January 23, 2015 ABSTRACT The expression of Piwi-interacting RNAs, small RNAs that bind to PIWI proteins, was until recently believed to be limited to germinal stem cells. We have studied the expression of PIWI genes during human lung embryogenesis and in paired tumor and normal tissue prospectively collected from 71 resected non-small-cell lung cancer patients. The mRNA expression analysis showed that PIWIL1 was highly expressed in 7-week embryos and downregulated during the subsequent weeks of development. PIWIL1 was expressed in 11 of the tumor samples but in none of the normal tissue samples. These results were validated by immunohistochemistry, showing faint cytoplasmic reactivity in the PIWIL1 -positive samples. Interestingly, the patients expressing PIWIL1 had a shorter time to relapse (TTR) ( p = 0.006) and overall survival (OS) ( p = 0.0076) than those without PIWIL1 expression. PIWIL2 and 4 were downregulated in tumor tissue in comparison to the normal tissue ( p < 0.001) and the patients with lower levels of PIWIL4 had shorter TTR ( p = 0.048) and OS ( p = 0.033). In the multivariate analysis, PIWIL1 expression emerged as an independent prognostic marker. Using 5-Aza-dC treatment and bisulfite sequencing, we observed that PIWIL1 expression could be regulated in part by methylation. Finally, an in silico study identified a stem-cell expression signature associated with PIWIL1 expression.

Surgery is the standard treatment for colorectal cancer (CRC), and adjuvant chemotherapy has been shown to be effective in stage III but less so in stage II. We have analyzed the expression of the miR-200 family in tissue samples from resected CRC patients and correlated our findings with survival to adjuvant treatment with fluoropyrimidines.Tumor tissue samples were obtained from 127 surgically resected patients with stage I-III CRC. miRNA detection was performed using TaqMan MicroRNA assays.High levels of miR-200a and miR-200c were associated with longer overall survival, while high levels of miR-429 correlated with longer overall and disease-free survival (DFS). In the subgroup of 56 patients treated with fluoropyrimidines and in the smaller subgroup of 32 stage II patients treated with fluoropyrimidines, those with high levels of miR-200a, miR-200c, miR-141, or miR-429 had significantly longer overall and DFS. Low miR-429 levels were identified as an independent prognostic marker. High levels of miR-429 combined with 5-fluorouracil inhibited cell invasion in LOVO cells.miR-200a, miR-200c, miR-141, and miR-429 expression levels may identify CRC patients, including those with stage II disease, who are most likely to benefit from adjuvant chemotherapy.

Introduction: Long non-coding RNAs (lncRNAs) have recently emerged as important actors in the regulation of multiple cellular processes including cancer. Acute myeloid leukemia (AML) is a heterogeneous disease; most of the main cytogenetic AML subgroups harbor a specific gene expression profile. AML with translocation t(8;16)(p11;p13) (t(8;16) AML) is a subtype with specific clinical and biological characteristics including a distinctive gene (Camós et al, Cancer Research 2006) and microRNA (Díaz-Beyá et al, Leukemia 2013) expression profile. In this translocation, MYST3 on chromosome 8p11 fuses with CREBBP on chromosome 16p13.3. The MYST3-CREBBP fusion protein is able to interact with multiple transcription factors (TF) producing a disturbed transcriptional program. However, the lncRNA expression pattern of different cytogenetic AML subtypes, including t(8;16) AML, have not been described yet. Aims: To examine the expression profile of lncRNAs within different AML subtypes, and to characterize the expression pattern of lncRNAs in t(8;16) AML in comparison to other AML subtypes. Patients and Methods: 46 AML patients, 4 normal bone marrow (NBM) and 3 CD34+ NBM samples were included in the study. Samples included different AML subtypes: intermediate-risk cytogenetic AML (IR-AML, n=18), t(15;17) (APL, n=4), t(8;21) AML (n=4), inv(16) AML(n=2), t(6;9) AML (n=7), AML with monosomal karyotype (n=4), t(3;3) AML (n=1), t(9;11) AML (n=1) and t(8;16) AML (n=5). Within IR-AML patients with a different mutational profile: FLT3-ITD (n=7), NPM1 (n=5), CEBPA (n=7) and DNMT3A (n=6) were included. The lncRNA expression was studied using Affymetrix® Human Gene 2.1 ST platform which includes 9698 lncRNAs transcripts. The filtering and normalization of the array data was performed using oligo package from Bioconductor. Statistical analyses were performed with TiGR MultiExperiment Viewer, BRB tools and R. The Transcription factor Affinity Prediction Web Tool was used to determine the putative transcription factors binding to the differentially expressed lncRNAs promoters. Results: The hierarchical cluster analysis showed that all 4 NBM as well as all 3 CD34+ NBM clustered together according to their lncRNA expression. Interestingly, all 5 t(8;16) AML samples clustered together, as well as the 3 APL, the 7 t(6;9) AML and 5 out of 7 cases with CEBPA mutations. The specific lncRNA signature of APL was composed of 79 differentially expressed lncRNA and t(6;9) AML lncRNA signature comprised of 15 differentially expressed lncRNAs. When we focused on t(8;16) AML lncRNA profile, we identified an specific 113-lncRNA signature in the supervised analysis (Figure). Interestingly, when we analyzed which (TF) had motifs overrepresented in the promoters regions of the t(8;16) AML lncRNA signature, we identified GATA2 as the TF with significantly overrepresented motifs for GATA2 (p Conclusions: LncRNAs expression profile seems specific of several AML subtypes, including t(8;16) AML. Some of the lncRNAs of this distinctive signature in t(8;16) AML are located in the HOXA genomic region, and correlate with several of the characteristic microRNAs previously described in this entity. Interestingly, we have predicted in silico GATA2, which interacts with CREBBP, as the most significant TF that could potentially regulate this lncRNAs signature. Nonetheless, further investigation is warranted to determine the mechanisms leading to this lncRNA signature and to identify the specific targets of these lncRNAs. Río Hortega CM13/00205, FIS PI13/00999 Disclosures No relevant conflicts of interest to declare.

11043 Background: Piwi-RNAs (piRNAs) are small non-coding RNAs (24-32 nt) required to protect the genome in germline cells. Initially they were thought to be expressed only in germline cells, but t...

Background: Non-coding RNAs (ncRNAs) have recently emerged as key regulators of diverse cellular processes, including leukemia. ncRNAs are classified according to their size as short (eg, microRNAs) or long ncRNAs. lincRNAs are long ncRNAs located in intergenic regions and have multiple regulatory functions, including gene expression regulation. Interestingly, active crosstalk between microRNAs and lincRNAs has been observed. lincRNAs are known to be deregulated in some cancers but their importance in acute myeloid leukemia (AML) is so far unknown. HOX genes play an important role in hematopoiesis and are deregulated in AML. lincRNAs are especially abundant in the clusters of HOX genes. HOTAIRM1, a myeloid lineage-specific lincRNA, is located at the 3’end of the HOXA cluster and seems to play a regulatory role in myelopoiesis. However, to date the potential prognostic role of HOTAIRM1 expression in AML has not been examined. Aims: To investigate first whether the expression of the lincRNA HOTAIRM1 is associated with the clinical, cytogenetic and molecular characteristics and microRNA expression in AML patients. Secondly, since intermediate risk (IR) AML patients have a highly diverse prognosis, we analyzed the potential prognostic value of HOTAIRM1 expression in IR-AML patients. Methods: To explore the expression level of HOTAIRM1 among different AML subtypes, we analyzed samples from 244 AML patients including CBF-rearranged AML (n=5), APL (n=4), MLL-rearranged AML (n=3), EVI1-rearranged AML (n=3), t(6;9) AML (n=9), AML with monosomal karyotype (n=3), and a large cohort of IR-AML (described below). For the analysis of prognostic value of HOTAIRM1, we analyzed specifically the outcome of 217 IR-AML patients (median age, 52; 51% males) sequentially included in CETLAM trials during the period 1995-2009. Molecular genotyping of this group identified NPM1 mutation (NPM1mut), FLT3-ITD, and biallelic CEBPA mutation (CEBPA mut) in 99 (45%), 79 (36%) and 17 (11%), respectively. The expression of HOTAIRM1 was analyzed using TaqMan® Gene Expression Assays (Applied Biosystems). microRNA and mRNA expression data were obtained in previous studies (Díaz-Beyá, Leukemia 2013). Statistical analyses were performed with BRB Array Tools, SPSS v20 and R v3.0. MaxStat package from R software was used to determine the optimal cutoff point of HOTAIRM1 expression. Results: Among all 244 patients, HOTAIRM1 expression was significantly different among the 7 included genetic subgroups (ANOVA p=0.0024), with the lowest levels observed in APL-AML patients and the highest in the t(6;9)AML patients. Within the IR-AML group, HOTAIRM1 overexpression was observed in NPM1mut patients (p The prognostic study showed that high HOTAIRM1 expression was associated with shorter 5-year overall survival (OS) (27+11% vs.47+8%; p=0.009) shorter 5-year disease-free survival (LFS) (22+12% vs. 53+9%; p Supervised analysis by means of t-test based on multiple permutations revealed a distinctive 33-microRNA signature which correlated with HOTAIRM1 expression including miR-196b (p Conclusion: The expression level of the lincRNA HOTAIRM1 varied among different molecularly-defined AML. Interestingly, HOTAIRM1 expression level showed independent prognostic value within the IR-AML group. Moreover, HOTAIRM1 expression strongly correlates with its neighboring HOXA4 gene and harbors a distinctive microRNA signature. Our findings can pave the way for further studies of HOX-related lincRNAs and microRNAs regulatory networks and their influence on clinical outcome. Acknowledgments: ISCIII RH13/00205, SEHH, FIS13/00999 Disclosures No relevant conflicts of interest to declare.

Introduction: PiwiRNAs (piRNAs) are small non-coding RNAs processed by PIWI proteins, a subfamily of Argonaute proteins. The main functions of piRNAs include regulation of chromatin and genome organization, DNA methylation, transposon repression and regulation of protein synthesis. Until recently, PIWI/piRNA functions were believed to be limited to germinal stem cells and early embryonic development, where they play a crucial role in the regulation of genomic stability. However, piRNA expression has now been identified in some tumors, although the specific prognostic impact of piRNAs remains to be elucidated. We have examined the potential reactivation of the PIWI/piRNA pathway in classical Hodgkin lymphoma (cHL) and its impact on prognosis in cHL patients. Methods: Formalin fixed and paraffin embedded tumor samples from 96 cHL patients were studied. All patients were diagnosed and treated in a single institution. HIV+ was an exclusion criterion. Median age was 33 years (range, 15–89); 47.9% were male; 30.2% were EBV+. The most frequent histological subtype was nodular sclerosis (65.6%). First-line treatment consisted of ABVD-type therapies in 90% of patients. 12 reactive lymph nodes (RLNs) were used as controls. The PIWI proteins PIWIL1 and PIWIL2 were studied by immunohistochemistry. The expression levels of three piRNAs (piR-651, piR-20365, piR-20582) were analyzed by Real-time PCR. cDNA was synthesized using miScript II RT Kit and Real-time PCR was performed using miScript SYBR Green PCR Kit (Qiagen). PiRNA expression was correlated with treatment response, disease-free survival (DFS) and overall survival (OS). Paired serum samples from seven prospectively collected cHL patients at diagnosis and at complete response and from 10 healthy controls were used to study the expression of piRNAs in serum. In addition, 5 cHL cell lines were used for the functional analysis. RNA hybrid, Renilla/Luciferase assay and Western Blot were used to identify and validate the piRNA targets. Statistical analyses were performed using R v2.13. Results: PIWIL1 and PIWIL2 proteins were expressed in the cytoplasm of the Hodgkin/Reed Sternberg (HRS) cells, indicating that the piRNA pathway is active in cHL. This was corroborated by the finding that the 3 piRNAs were also expressed in all tumor samples and in the 5 cHL cell lines. piR-651 (p In situ hybridization with LNA probes (Exiqon) showed that piR-651 was highly expressed in the cytoplasm of HRS cells. We studied putative targets genes of piR-651 using RNA hybrid. We identified several candidate genes related to drug metabolism, including ABCC5, an efflux pump of multiple drugs including doxorubicin. We cloned the 3’UTR region of ABCC5 in the psicheck2 vector and performed a Renilla/Luciferase assay. We observed a significant increase of 28% (p=0.047) in the Renilla luciferase levels in the cells transfected with 200nM of piR-651 inhibitor compared to oligo control. To further validate this, we transfected 3 HL cell lines with piR-651 inhibitor or oligo control and analyzed the protein levels of ABCC5 by Western Blot, observing a mean increase of 18% in the cells transfected with piR-651 inhibitor. These results showed that piR-651 inhibits the translation of ABCC5. Conclusions:The PIWI/piRNA pathway is reactivated in cHL and the expression of piRNAs affects tumorogenesis and cHL patient outcome. piR-651 influences treatment response and survival through regulation of ABCC5. Our findings provide the groundwork for further investigation of the novel role of piRNAs in HL. Acknowledgments : AECC-Catalunya (2014), APIF-UB (AC). Disclosures No relevant conflicts of interest to declare.

Introduction The behavior of classical Hodgkin lymphoma (cHL) is determined by both the intrinsic features of the Hodgkin/Reed Sternberg (HRS) cells and the characteristics of the microenvironment, making the analysis of entire lymph nodes an effective approach to understanding the disease. One of the genetic lesions most frequently found in HRS cells involves members of the JAK/STAT signaling pathway. However, few studies have identified microRNAs (miRNAs) that target JAK2 in cHL. In a previous study, our group identified miR-135a as a JAK2 regulator and showed the prognostic impact of this miRNA in a small set (n=89) of cHL patients (Navarro A. et al. Blood 2009). In the present study, we have examined novel miRNAs targeting JAK2 and evaluated the prognostic impact of their expression levels in 170 lymph nodes of cHL patients. Methods TargetScan and miRbase were used to identify JAK2-related miRNAs. The conserved miRNAs with higher scores were selected and further validated by Renilla-Luciferase assay and Western Blot. We performed a Renilla-Luciferase assay with a modified expression vector psiChecK-2 containing the complete 3’UTR region of JAK2 cloned in the 3’UTR region of Renilla luciferase gene. For Renilla-Luciferase assay, 100nM of the pre-miRNAs of interest or pre-miR negative control were transfected in the HDMYZ cHL cell line together with 1μM of modified psicheck2 vector using Lipofectamine 2000, and luminescence was read at 24 hours. Further validation of the miRNA target was performed by Western Blot in three cHL cell lines (L428, L-1236 and HDMYZ). The expression levels of the miRNAs identified as targeting JAK2 were then assessed in lymph nodes from 170 cHL patients (median age, 33 years; male, 51%; EBV, 43.8%; HIV, 12%) diagnosed and treated in a single institution. Moreover, 15 reactive lymph nodes (RLN) were used as normal controls. RNA was purified from formalin-fixed paraffin-embedded lymph nodes using RecoverAll Total Nucleic Acid Isolation kit. The miRNA expression was analyzed using TaqMan microRNA assays. Statistical analyses were performed using R v2.13 and PASW Statistics 18. Results The bioinformatic analysis identified seven microRNAs as possible regulators of JAK2 (miR-34a, miR-101, miR-135a, miR-204, miR-216a, miR-216b and miR-375). The Renilla-Luciferase assay confirmed that in addition to miR-135a, miR-101 (61%; p=0.01), miR-204 (46%; p=0.003) and miR-216a (64.8%; p=0.02) also targeted JAK2. These findings were confirmed by Western Blot analysis of JAK2 levels after transfection with pre-miRNAs (median % of protein reduction in the 3 cell lines of 21.1%, 46.7% and 24%, respectively). The analysis of these miRNAs in RLN showed that miR-101, miR-135a and miR-204 were significantly downregulated in lymph nodes of cHL patients (p Conclusions MiR-101, miR-135a, miR-204 and miR-216 regulate JAK2 and are independent prognostic factors in cHL. Acknowledgments SDCSD of School of Medicine of UB. AC is an APIF-UB fellow. Disclosures: No relevant conflicts of interest to declare.

Long non-coding RNAs (lncRNAs) are deregulated in several tumors, although their role in acute myeloid leukemia (AML) is mostly unknown. We have examined the expression of the lncRNA HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) in 241 AML patients. We have correlated HOTAIRM1 expression with a miRNA expression profile. We have also analyzed the prognostic value of HOTAIRM1 expression in 215 intermediate-risk AML (IR-AML) patients. The lowest expression level was observed in acute promyelocytic leukemia (P < 0.001) and the highest in t(6; 9) AML (P = 0.005). In 215 IR-AML patients, high HOTAIRM1 expression was independently associated with shorter overall survival OR: 2.04; P = 0.001), shorter leukemia-free survival (OR: 2.56; P < 0.001) and a higher cumulative incidence of relapse (OR: 1.67; P = 0.046). Moreover, HOTAIRM1 maintained its independent prognostic value within the favorable molecular subgroup (OR: 3.43; P = 0.009). Interestingly, HOTAIRM1 was overexpressed in NPM1-mutated AML (P < 0.001) and within this group retained its prognostic value (OR: 2.21; P = 0.01). Moreover, HOTAIRM1 expression was associated with a specific 33- microRNA signature that included miR-196b (P < 0.001). miR-196b is located in the HOX genomic region and has previously been reported to have an independent prognostic value in AML. miR-196b and HOTAIRM1 in combination as a prognostic factor can classify patients as high-, intermediate-, or low-risk (5-year OS: 24% vs 42% vs 70%; P = 0.004). Determination of HOTAIRM1 level at diagnosis provided relevant prognostic information in IR-AML and allowed refinement of risk stratification based on common molecular markers. The prognostic information provided by HOTAIRM1 was strengthened when combined with miR-196b expression. Furthermore, HOTAIRM1 correlated with a 33-miRNA signature., Marina Diaz-Beya is supported by ISCII (Rio Hortega CM13/00205). This research was in part supported by Fundacion Espanola de Hematologia Hemoterapia (beca de investigacion MDB). This research is also supported by grants from Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III PI13/00999 (IP: Dr. Jordi Esteve), RETICS RD12/0036/0010 (JE; MDB) and SDCSD from School of Medicine, University of Barcelona, AECC-Catalunya 2013 (AN) (sponsored by Mat Holding). Anna Cordeiro is an APIF fellow of the University of Barcelona