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Abstract 3252 Poster Board III-1 A better understanding of the mechanisms which regulate drug resistance is critical for improving therapy for patients at risk of poor response. Even for CML patients the long-term benefits of the treatment are limited by the emergence of resistance, although the therapy has been dramatically improved by Imatinib Mesylate, which inhibits Bcr-Abl activity. One of the most important mechanisms of resistance is the disregulation of various members of the highly conserved family of transmembrane proteins characterized by an ATP-binding cassette domain, called ABC superfamily of transporters (Dean et al., Genome Res 2001). In CML cells an aberrant expression of ABC transporter genes has been described to be mediated by specific transcription factors, which are in turn affected by an aberrant activity of Bcr-Abl. It has been shown that Bcr-Abl can indirectly activate c-Myc via the JAK2 pathway, which increases a translation of c-Myc mRNA. In addition, in a subgroup of CML patients a chromosome 8 trysomy, has been observed, which can be associated with c-Myc amplification. In this study we have investigated whether c-Myc can regulate transcription of ABC tranporter genes in CML. Initially, ABC transporter gene expression has been monitored in HL60, a human promyelocytic cell line in which c-MYC is overexpressed. We have examined the expression level of all 48 human ABCs transporters as a function of c-MYC silencing. Our results have demonstrated that c-Myc regulates the transcription of several ABC genes, such as ABCA2, ABCB9, ABCB10, ABCC1, ABCC4, ABCE1, ABCF1, ABCF2, a majority of which has been found implicated in drug resistance. Furthermore, by performing chromatin immunoprecipitation (ChIP) we have shown a direct binding of c-Myc to the promoters of those ABC transporter genes in HL60 cell line. In addition, by ChIP we have demonstrated that c-MYC is physically bound to the promoter of tested ABC genes in CML cell lines KG-1a and K562. Based on those findings we have investigated the expression level of c-Myc in CD34+ progenitor cells derived from CML patients. Also, we have evaluated the effects of highly expressed c-Myc on ABC transporters. Our results have shown in a group of 20 newly diagnosed chronic phase (CP)-CML patients an increased expression of c-Myc in CD34+ cell fraction, when compared to the expression level of c-Myc in the entire population of mononuclear cells from which CD34+ cell fraction has been purified. In those cells we have identified in association with an increased level of c-Myc an increased expression of the same subset of ABC genes, which we have observed in cell lines. Furthermore, we have evaluated whether a differential expression of c-Myc and ABC transporter genes is associated with low Sokal risk and high Sokal risk patients. This analysis has been performed on CD34+ cells purified from 19 CML patients of whom 12 were scored as low Sokal risk and 7 as high Sokal risk. Our results have shown that expression of c-Myc and ABCC4 is different in those two patients population (p Disclosures: No relevant conflicts of interest to declare.

Abstract 854 It has been proposed that most protein-encoding genes may be regulated by small multifunctional RNAs which can control transcript turnover and/or protein translation (siRNA and miRNA). Specifically, miRNAs have been shown to act predominantly at the level of translation by blocking the access or sliding of ribosomes to mRNAs (3'UTR). Several studies have shown that multiple miRNAs can be disregulated in tumours as compared to normal tissues. For example, the miR-17-92 cluster is up regulated in CML, suggesting their involvement in leukemiagenesis (Venturini et al., Blood 2007). However, a role of miRNA in preventing tumor development and progression has also been suggested. Bueno et al have shown that hsa-miR-203 can specifically target BCR-ABL and reduce its expression in CML derived cell lines (Bueno et al., Cancer Cell 2008). Since multiple miRNAs seem to act in a combinatorial fashion to regulate mRNA translation, we hypothesised that other miRNAs in addition to hsa-miR-203 might be involved in BCR-ABL expression control. To test this hypothesis we conducted a search for miRNAs specifically targeting Bcr-Abl, using the miRBASE program to scan human genome (http://microrna.sanger.ac.uk/). This search identified 15 miRNAs potentially able to target the BCR-ABL 3' UTR. Further investigation showed that only hsa-miR-451, hsa-miR-515-3p and hsa-miR-760 had a sufficiently high score to predict genuine interactions with the 3'UTR of BCR-ABL and therefore only these miRNAs were utilized for further analyses. Initially, the three miRNAs were transfected in K562 Ph+ cells, together with hsa-miR-203 as a positive control and a scrambled miRNA used as a negative control. BCR-ABL expression was monitored at both mRNA ( qRT-PCR) and protein level (Western Blotting). Our results demonstrated that BCR-ABL mRNA expression was unaffected by miRNAs, whereas protein expression was significantly reduced. Considering the combinatorial function of miRNAs on their target mRNAs, we also tested whether a pool of the four identified miRNAs could be effective in suppressing BCR-ABL expression. In those experiments, the molar concentration of each miRNA was a quarter of that used for a single miRNA transfection. The results have shown that the pool of miRNAs worked efficiently in suppressing Bcr-Abl expression, which suggested a cooperative function of the four miRNAs in controlling both the expression and translation of Bcr-Abl. To further confirm that miRNAs directly targeted BCR-ABL, the 3'UTR of the gene was cloned downstream of a reporter renilla luciferase gene. The reporter was co-transfected in HEK293 cells together with single miRNAs or a pool of them, and luciferase activity was quantified. Those results show that the presence of each miRNAs significantly reduced the luciferase activity as compared to that obtained by transfecting cells with a scrambled miRNA. These experiments therefore confirmed a direct effect of the four miRNAs on the 3'UTR of BCR-ABL. Again the “pool” of miRNAs showed the strongest effect on the luc reporter expression. Through an additional deletion analysis we mapped the regions of 3'UTR of BCR-ABL targeted by the four miRNAs, in order to confirm that the predicted binding regions of miRNAs are critical to mediate repression of BCR-ABL. In conclusion, our study identified three new human miRNAs having a potential to specifically target BCR-ABL and suppress its translation and expression in CML cells. BCR-ABL, which plays a critical role in CML, is effectively suppressed by TK inhibitors, exemplified by Gleevec and our findings provide a rationale to exploit miRNA as an alternative therapeutic approache which could further improve CML treatment, or to complement TK inhibitors in an effort to eradicate minimal residual disease. Supported by: Novartis Oncology, Clinical Development, TOPS Clinical Correlative Studies Network Disclosures: No relevant conflicts of interest to declare.

We describe the purification and cloning of human DNA replicated at the onset of S phase in HL60 cells synchronized with aphidicolin. A survey of the overall structural properties of these sequences did not show any distinctive features except for an enrichment in Cot0 DNA. The two longer fragments were completely sequenced and studied in more detail. Both were shown to contain transcriptional signals associated with promoters and/or enhancers, such as the binding sites of Sp1, T antigen and nuclear factor III. In one instance, a binding site for a known cellular transcription factor (USF/MLTF) was located inside the sequence by footprinting. Accordingly, by CAT assay and Northern blot, the same sequence was shown to contain an active promoter. The significance of these findings with respect to the role of transcription in initiation of DNA replication at the origin is discussed. None of the tested fragments exhibited autonomously replicating sequence (ARS) activity in transfected cells. The problems connected with the detection of ARS activity in human cells are critically examined.

// Diana T. Lau 1 , Claudia L. Flemming 1 , Samuele Gherardi 2 , Giovanni Perini 2 , Andre Oberthuer 3 , Matthias Fischer 3 , Dilafruz Juraeva 4 , Benedikt Brors 4 , Chengyuan Xue 1 , Murray D. Norris 1 , Glenn M. Marshall 1,5 , Michelle Haber 1 , Jamie I. Fletcher 1,* and Lesley J. Ashton 6,* 1 Children’s Cancer Institute Australia, Lowy Cancer Research Centre, Randwick, NSW, Australia 2 Department of Biology, University of Bologna, Bologna, Italy 3 Children’s Hospital, Department of Pediatric Oncology and Hematology, University of Cologne and Centre for Molecular Medicine Cologne, Cologne, Germany 4 Division of Theoretical Bioinformatics, German Cancer Research Center, Heidelberg, Germany 5 Kids Cancer Centre, Sydney Children’s Hospital, Randwick, NSW, Australia 6 Faculty of Medicine, School of Women’s and Children’s Health, University of New South Wales, Sydney, NSW, Australia and Research Portfolio, University of Sydney, Sydney, NSW, Australia * These authors have contributed equally to this work Correspondence to: Jamie I. Fletcher, email: // Lesley J. Ashton, email: // Keywords : MYCN, MYC, SLC19A1, methotrexate, neuroblastoma Received : February 10, 2015 Accepted : March 10, 2015 Published : March 30, 2015 Abstract MYCN amplification occurs in 20% of neuroblastomas and is strongly related to poor clinical outcome. We have identified folate-mediated one-carbon metabolism as highly upregulated in neuroblastoma tumors with MYCN amplification and have validated this finding experimentally by showing that MYCN amplified neuroblastoma cell lines have a higher requirement for folate and are significantly more sensitive to the antifolate methotrexate than cell lines without MYCN amplification. We have demonstrated that methotrexate uptake in neuroblastoma cells is mediated principally by the reduced folate carrier (RFC; SLC19A1 ), that SLC19A1 and MYCN expression are highly correlated in both patient tumors and cell lines, and that SLC19A1 is a direct transcriptional target of N-Myc. Finally, we assessed the relationship between SLC19A1 expression and patient survival in two independent primary tumor cohorts and found that SLC19A1 expression was associated with increased risk of relapse or death, and that SLC19A1 expression retained prognostic significance independent of age, disease stage and MYCN amplification. This study adds upregulation of folate-mediated one-carbon metabolism to the known consequences of MYCN amplification, and suggests that this pathway might be targeted in poor outcome tumors with MYCN amplification and high SLC19A1 expression.

The deregulation of ATP-binding cassette (ABC) transporters responsible for the efflux of anticancer agents may involve mutations or single nucleotide polymorphisms (SNPs) or an increase in their expression level. Consequently, chemoresistance may develop. We have previously shown that the expression level and transcription of ABC drug transporters in CML cells is affected by c-Myc. Our results demonstrated that c-Myc is highly expressed in CD34+ cells from newly diagnosed chronic phase (CP)-CML patients, and that it can significantly upregulate the expression of several ABC genes, particularly, the ABCC1, ABCC4 and ABCG2, while it downregulates the expression of ABCC3. We have also demonstrated that c-Myc was a direct regulator and physically associated with the promoter of tested ABC genes, as assessed by Chromatin Immunoprecipitation in a cell line derived from a Ph+ CML patient. Taken together, our findings supported the model of a direct and coordinate regulation of a large set of ABC genes by the c-Myc transcription factor. Our study also supported prior findings that deregulation of specific set of ABC genes could be an important molecular mechanism altering imatinib transport. Based on these observations we have started to investigate the role of ABC transport genes expression in newly diagnosed CP-CML patients treated with imatinib. RNA extracted from white blood cells of 5 patients who achieved a stable major molecular response (MMR) by 12 months (responders) and 15 patients who didn’t show a partial cytogenetic response (CgR) by 6 months nor a complete CgR by 12 months (suboptimal responders according to European LeukemiaNet recommendations). All pts were enrolled on GIMEMA CML Working Party-sponsored clinical trials of imatinib. A panel of ABC genes including ABCB1, ABCB9, ABCC1, ABCC3, ABCC4, ABCE1 and ABCG2 was interrogated by Q-PCR for the level of expression. Results were normalized to the expression of three reference genes, i.e., GUSB, b-actin and GAPDH. Our results show that suboptimal responders display high expression levels of ABCG2 (p

Our findings support a model in which the GSK3 pathway can exerts a negative control on p75NTR transcription in high risk neuroblastoma and highlight the possibility to use GSK3 inhibitors as potential therapeutic drugs to treat neuroblastoma with low p75NTR.