ALBERT

Description: Key Points There is 100% concordance in the cytogenetic and mutation profile between PB and BM in myelodysplastic syndrome.

Description: Abstract 3874 MicroRNAs are a class of small RNA molecules that regulate numerous critical cellular processes including proliferation, differentiation and apoptosis. Several microRNAs play important roles in normal haematopoiesis such as mir-181 which inhibits differentiation, mir-223 induces myeloid differentiation and mir-150 and mir-155 that are involved in T and B cell differentiation. Increased levels of mir-155 and mir-181 have been documented in diffuse large B-cell lymphomas and acute myeloid leukaemia respectively and mir-15a and mir-16 are frequently deleted in chronic lymphocytic leukaemia. More recently, down-regulation of mir-145 and mir-146a, located to the critical deleted region of 5q in MDS 5q- syndrome has been implicated in this disease. To obtain insight into the function of miRNAs, much effort has gone into different computational algorithms that identify miRNA targets. However, a major drawback of these prediction models is the substantial false positive rate and an inevitable bias due to reliance on the few known miRNA:target gene interactions. The lack of sensitivity and specificity of the developed computational algorithms is clearly shown by the fact that for the 940 human miRNAs identified (miRecords release 10 April 2010), only 152 miRNAs have experimentally validated targets. There is thus, a clear need to develop methodologies for the identification and validation of the functional targets of specific miRNAs. To enable the identification of biologically relevant microRNA targets we have developed a novel functional assay for the isolation of microRNA target sequences by selection, relying directly on downregulation, by a miRNA, of a selectable marker expressed in frame with a library of 3′ RNA sequences. The library was derived from human brain tissue cDNA, brain being the tissue expressing the largest number of individual genes (∼11,000). Cells with low or absent levels of the miRNA of interest are transfected with this 3′ UTR library inserted downstream of a TKzeo fusion gene in plasmid p3′TKzeo. Zeocin selection results in a population of cells that are expressing the TKzeo fusion protein and are resistant to zeocin and sensitive to Ganciclovir (GCV). The zeocin resistant cells are next transfected with the miRNA of interest cloned into the pbabepuro expression vector and selected in puromycin to isolate microRNA transduced cells. GCV treatment then selects for cells that have downregulated the TKzeo fusion protein expression either by inhibition of translation or mRNA cleavage. The 3′UTR sequences present downstream of the TKzeo fusion from GCV resistant cells are PCR amplified and sequenced. As proof of concept we identified targets for mir-130a which is involved in megakaryopoeisis and for which validated targets have been identified. This microRNA is not expressed in MCF7 cells, therefore the library was introduced into MCF7 cells and selected in 500μg/ml zeocin. Introduction of mir-130a, GCV selection, PCR amplification, cloning and sequencing of the introduced 3′ UTR sequences resulted in the identification of musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB), a known validated target for mir-130a. In addition, we identified tumour protein translationally-controlled 1 (TPT1), proline rich 14 (PRR14), kinesin-associated protein 3 (KIFAP3), microtubule interacting and transport, domain containing 1 (MITD1) and cytochrome P450, family 27, subfamily A, polypeptide 1 (CYP27A1). All targets were validated by western blot analysis of cell extract from cells over-expressing mir-130a or cells treated with hairpin inhibitors directed against mir-130a. Deep sequencing of the total PCR product identified 107 putative targets for mir-130a which are being verified by western blot analysis of the genes for which antibodies are available. This strategy makes no assumptions based on previously identified sequences, relying directly on downregulation, by a miRNA, of a selectable marker expressed in frame with a library of 3′ RNA sequences. This strategy will lead to identification of functional targets for all the majority of microRNAs. For example the microRNAs present on the critical deleted chromosomal region of a number of haematological malignancies, including those on chromosomes 5 and 7 such as mir-143, mir-145, mir-146a, mir-378 etc. Disclosures: Gaken: Sigma: Patents & Royalties. Mohamedali:sigma: Patents & Royalties.

Description: Introduction: The importance of miRNAs in regulating gene expression has been addressed in several haematological cancers including myelodysplastic syndrome (MDS). In MDS, miR-145 and miR-146a have been associated with some of phenotypic features of 5q- syndrome. Monosomy 7/7q deletion is the second most common chromosomal abnormality in MDS and identifies a subgroup of patients with poor prognosis. Thus far, no studies have examined the role of miRNA dysregulation in MDS patients with monosomy 7. In this study we examined the functional consequences of deletion of miRNAs localized to 7q with particular focus on miR-595, which is localized to 7q36.3, which is commonly deleted region in 7q- and -7. Most of the available methodologies for miRNA target identification are based on computational algorithms. Therefore, there is a relatively high incidence of false positive target identification. To enable the identification of biologically relevant microRNA targets, a novel functional assay was developed in our lab, which is based on positive/negative selection (Gaken et al, 2012). Using this assay, we identified functional target of miR-595 and correlated this with the MDS phenotype. Method: The functional assay relies on miR-595, identified several targets including RPL27A, HSPA14, GRM5 and SEC63. We focused on ascertaining the biological function of RPL27A. RPL27A was validated as a target for miR-595, by examining the changes on RPL27A expression using quantitative RT-PCR and western blot analysis in cells transfected with plasmid expressing miR-595. We demonstrated a targeted reduction of RPL27A in several cell lines including HCT-116 and HEL cells (expressing p53) as well as HCT-116 p53-/- and K562 cells (not expressing p53) through retroviral expression of (shRNAs). RPS14 knocked down was used as a control. The knockdown of RPL27A and RPS14 was also performed in normal CD34+ (n=6) in two stages liquid culture using cytokines mixture to induce erythroid, myeloid and megakaryocyte differentiation. The effects on expressing erythroid cells were assessed and composed of CD71 and GlyA, myeloid and megakaryocyte cells using CD11b and CD41, respectively. Result: RPL27A was validated as target for miR-595 (Figure1). RPL27A knockdown resulted in a reduction in the large ribosomal subunit (60S) based on Polysomes fractionation, (Figure 2). On day 6 of the knockdown, the depletion of RPL27A led to p53 activation and induced apoptosis in all cell lines at different levels, 40-60% increases in apoptotic cells in HCT-116 and HEL cell lines and 20-30% increases in apoptotic cells in HCT-116 p53-/- and K562 cells (Figure 3). Interestingly, like RPS14 knockdown, the effect of RPL27A knockdown in 48 h was p53 dependent. The p53 independent effect, which was observed on day 6, was attributed to a defect in ribosome biogenesis based on the disruption of nucleolar staining in HCT-116 and HCT-116 p53-/- with depleted RPL27A using fibrillarin antibody and similar effects were seen with RPS14. To investigate whether the effect of RPL27A knockdown on erythroid differentiation is similar to the other ribosomal proteins, RPL27A knockdown (approximately 70% reduction at day 6) was performed in normal CD34+ and resulted in reduce viability and apoptosis within 10 days. Post-transcriptional p53 overexpression was also observed; this was associated with a marked increase in the mRNA expression of p53 targets, p21 and Bax. Following 10 days in liquid culture, RPL27A knockdown blocked the proliferation and differentiation of erythroid cells relative to myeloid and megakaryocyte lineages. Similar observations have been made in 5q- syndrome due to RPS14 haploinsufficiency and in DBA patients with mutations in large or small ribosomal subunit proteins. Conclusion: This study showed that haploinsufficency of miR-595 in patients with -7/7q del is important in pathogenesis of reduced erythropoiesis and a reduction in myeloid and erythroid progenitor populations. The effects are mediated via RPL27A, which is a ribosomal protein and its knockdown produces it effects via p53 dependent pathways, leads to ineffective erythropoiesis and arrest of the myeloid and megakaryocyte differentiation. These effects are particularly relevant in patients with complex chromosomal abnormalities that include haploinsufficiency of RPS14 due to -5/del 5q. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 683 Introduction: FL is one of the commonest B-cell lymphomas accounting for 25–30% of all newcases of non-Hodgkin's lymphoma. Median survival ranges from 6–10 years with a constant annual rate of relapse and death. The cellular immune system through T-cells and macrophages/monocytes (MMs) has an important role in the response to therapy and long term remissions. Evidence for this stemmed from increased incidence and the poor response rates and survival in patients with inherited or acquired T-cell defects, and the graft-versus-leukaemia effect of allogeneic stem cell transplants. The number of tumour infiltrating lymphocytes (TILs) and MMs appear to predict clinical response and outcome in FL. A recent National Cancer Institute study looked at the prediction of survival in FL based on the molecular features of TILs with positive findings. However, this study used frozen material which could have changed the signatures, T-cells were not positively selected and would have been mixed with other non B-cells, negatively selected cells were stimulated with CD3 which could have changed the gene expression profile, the study looked only at the predictive value in relation to prognosis and did not look at PB T-cell (PBT)profile. Aims and methods: In the first stage reported here, we looked at positively selected TILs, PBT and MMs gene expression profile signatures in fresh lymph node samples (LNs) of histologically confirmed grades 1–3 FL in 14 patients and compared the profile results with similar cells positively selected from PB samples taken at the same time. The analysis was controlled with 4 histologically proven reactive hyperplasia LNs. Tissue samples were digested with a cocktail of enzymes, ficol separated and then positively selected for CD2, CD19, CD14 in that order using Dynabeads (Dynall) initially then Microbeads (Miltenyl Biotech) subsequently. CD2 was used to avoid T-cell receptor stimulation and change of expression with CD3. Some isolated T-cells were initially expanded in culture for purity assessment only by flow cytometry. T-cells and MMs were liquid nitrogen frozen in trisol whilst B-cells were stored in DMSO. RNA was extracted at the same time and analysed using Affymetrix Microarray Chips. Statistical analysis used 3 parameters to define significant change in expression: fold change of 〉1.5, p value of 〉 1×10−3and FDR of 〉0.5. Samples were analysed as paired and pooled as some cases did not yield adequate paired samples. Results: T-cell paired (10) samples showed 97 over (41) or under (56) expressed genes in TILs compared to PBT whilst pooled samples (14) showed 778 over (380) or under (398) expressed genes. The top over expressed genes (10 fold plus) using both methods of analysis were CXCL13 a B-cell chemotactic gene, IGJ which helps in assembling IgG and IgM, CTLA4 a regulatory molecule, CD200 which delivers an inhibitory signal for the macrophage lineage and several heat shock protein (HSP) genes. The top under-expressed genes (10 fold plus) include genes that regulate T-cell adhesion, migration and direct and indirect cytotoxicity such as a number of FC receptor genes needed for antibody dependant cell cytotoxicity, killer cell lectin-like receptor genes, fractalkine genes involved in the adhesion and migration of leukocytes, and granzyme, granulosin, lysosyme and other genes involved in T-cell cytotoxocity. MMs: Only pooled samples were analysed as the extracted cell numbers were too small to have paired analysis. Many more (3239) genes were over (1494) or under (1745) expressed in LN MMs compared to PB MMs. The top over expressed genes include genes that support B cell growth such as IGJ, BLNK and C4orf7, the B cell chemotactic gene CXCL13, signal transduction inhibitors and HSPs. The top underexpressed genes include those that regulate cell adhesion and survival, cell proliferation and migration and extracellular matrix assembly. Others include FC receptor, IL-1 and compliment genes necessary for cell cytotoxicity, and thrombomodulin genes. Conclusion: We believe this is novel data suggesting that TILs and tissue MMs appear to support B-cell growth in FL whilst inheriting several defects in direct and indirect cell cytotoxicity. Downregulating the significantly overexpressed and upregulating the under expressed genes through targeted therapy may provide the basis of non-chemotherapy immunomodulatory treatment of B-cell lymphomas. Disclosures: Rassam: BMS: Honoraria; Johnson and Johnson: Unrestricted research grant Other; ROCHE: Honoraria. Mufti:Celgene: Consultancy, Research Funding.

Description: Introduction: Tumour infiltrating lymphocytes (TIL), tissue macrophages/monocytes (TMM) and follicular dendritic cells (FDC) have an important role in the biology of FL. Previous GEP studies used stored material, non-trizol freezing, negative T cell selection and CD3 stimulation and culture which could change signatures. None looked at peripheral blood (PB) T-cell (PBT) or MM (PBMM) profiles concurrently or specifically at fresh MM signatures though numbers are implicated in the disease's biology. Aims and methods: In the first stage we looked at positively selected TIL and TMM gene expression profile signatures in fresh diagnostic samples of histologically confirmed grades 1-3a FL in 14 patients and compared the profiles with similarly selected cells from controls and PB samples taken at the same time. We positively selected for CD2, CD14 and CD19 in that order with microbead technology. CD2 selection with no expansion in culture were used to minimise T-cell receptor stimulation. Cells were liquidised in Trizol prior to freezing. RNA was extracted and analysed using Affymetrix Microarray Chips. Statistical analysis used 3 parameters to define significant change in expression: fold change of 〉1.5, p value of 〉 1x10-3 and FDR of