ALBERT

Description: Abstract 2891 Thiopurines; mercaptopurine (6-MP) and 6-thioguanine (6-TG) are important drugs in treatment of paediatric cancer patients. The activity of these drugs depends on the activity of several common enzymes in the metabolism pathways such as thiopurine methyl transferase (TPMT) and guanine monphosphate synthetase (GMPS). In the present study the efficacy of thiopurines was investigated upon inhibition of TPMT and GMPS gene expression by RNA interference (siRNA). Treatment of the MOLT4 human T-cell leukemia cells with TPMT and GMPS siRNA resulted in decreased mRNA expression as determined by Real-Time PCR by 60% and 70% respectively. When reducing TPMT mRNA, the MOLT-4 cells were 70% less sensitive to 6-MP while the sensitivity to 6-TG was unchanged. When down-regulating GMPS using siRNA the sensitivity was unchanged upon treatment with both drugs. A microarray experiment was conducted using wild type on MOLT-4, 6-MP and 6-TG resistant MOLT-4 variants. The aim was to identify affected genes in response to the resistance induction. The mRNA levels of several nucleoside transporter genes were down-regulated in both thiopurine-resistant sub-lines; concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2). The expression of genes encoding the purine de novo synthesis enzymes was reduced to the same extent in both resistant cell lines as well as the expression of GMPS (〉40% in both resistant sub-lines) which indicates defected purine metabolism. The activity of TPMT and other enzymes in metabolism pathway of 6-MP and 6-TG was unchanged in resistant cells. Our results suggest that the contribution of TPMT activity in cytotoxicity of 6-MP is greater than for 6-TG, probably due to inhibition of de novo purine synthesis by methylated nucleotides, whereas TPMT inactivates 6-TG by methylation. Disclosures: No relevant conflicts of interest to declare.

Description: Our group previously identified two novel genes, RFP2/LEU5 and DLEU2, within a 13q14.3 genomic region of loss seen in various malignancies, including chronic lymphocytic leukemia and mantle cell lymphoma. However, no specific inactivating mutations were found in these or other genes in the vicinity of the deletion, strongly suggesting that a nonclassical tumor-suppressor mechanism may be involved. Our recent analysis demonstrates that the DLEU2 gene encodes a putative noncoding antisense RNA, with one exon directly overlapping the first exon of the RFP2/LEU5 gene in the opposite orientation. In addition, the RFP2/LEU5 transcript can be alternatively spliced to produce either several monocistronic transcripts or a putative bicistronic transcript encoding two separate open-reading frames, adding to the complexity of the locus. The finding that these gene structures are conserved in mouse, including the putative bicistronic RFP2/LEU5 transcript as well as the antisense relationship with DLEU2, further underlines the significance of this unusual organization and suggests a biological function for DLEU2 in the regulation of RFP2/LEU5. Further characterisation of the 13q14 deletion locus distal to the RFP2/DLEU2 region has resulted in the identification of two novel evolutionary conserved genes DLEU7 and DLEU8, the latter of which may function as both a protein encoding and an cis-antisense gene. The combined data indicates that the CLL 13q14 deletion locus encompasses a highly complex gene regulatory system, the partial deletion of which may affect the post-transcriptional regulation of non-deleted genes in the vicinity.