Mechanism of Resistance to Azacitidine in Myelodisplastic Syndromes.
Authors: Martinez Ana Belen Valencia , Martino Alessandro , Buchi Francesca , Masala Erico , Sanna Alessandro , Canzian Federico , Cilloni Daniela , Gaidano Valentina , Gozzini Antonella , Bosi Alberto , Santini Valeria 
Published: October 2019

Abstract
Abstract 2810
Azacitidine is a pyrimidine nucleoside analogue of cytidine and it is at present the standard treatment for myelodysplastic syndromes (MDS). A large number of studies performed to evaluate the mechanism of resistance/response to nucleoside chemotherapeutics like cytosine arabinoside or gemcitabine, have demonstrated the importance of the levels of expression of the enzymes involved in their metabolism. Despite the high clinical interest in azacitidine, little is known about its intracellular metabolism and scanty data are available in literature. Recently, differences in the expression of these enzymes has been reported in cell lines differentially sensitive to hypomethylating agents. Clinical responses to azacitidine are heterogeneous, mainly due to clinical characteristics of MDS patients, so there is a need to identify molecular markers that could predict and/or monitor the efficacy of this therapy. A better understanding of the biological mechanisms involved in the activation and DNA uptake of azacitidine is therefore necessary to possibly identify responsive MDS patients.

Objectives:
To measure the expression levels of the 5 enzymes involved in azacitidine metabolism and to correlate their gene expression with the clinical response to the drug. To test possible causes of differences in gene expression.

Material and Methods:
DNA and RNA was extracted from mononuclear cells of 39 IPSS high risk MDS patients treated with at least 6 cycles of azacitidine 75mg/m2/7 days every 28 days. Of them, 14 patients were responders and 25 non-responders to azacitidine according to IWG 2006 criteria. Gene expression was assessed with quantitative PCR, using an ABI GeneAmp® 5700. The specific oligonucleotides and TaqMan® probes of every selected gene will be acquired among the Assay-on-Demand® Gene Expression Products (Applied Biosystems). Promoter methylation of UCK1 was evaluated by methylation specific PCR using specific primers. The UCK1 coding region was sequenced by Sanger method.

Results:
Gene expression of hENT1, UCK1, DCK, RNR1 and RNR2 was compared between the groups of responders and non-responders. Individually, none of the genes was differentially expressed in responders versus non-responders. Nevertheless, a trend for lower expression in non-responders was shown for the UCK1 gene (median responders: 12.4 versus median non responders: 10.7; P = 0.095). In order to verify whether the presence of aberrant methylation could impact in UCK1 gene expression, we measured the methylation status of a putative CpG island in the UCK1 gene promoter region. The CpG island was unmethylated in all the samples analyzed, including responders and non-responder cases, as well as healthy donors. Moreover, to verify whether the difference in gene expression could be due to nucleotide variants in the gene sequence, we sequenced the UCK1 sequence. We found one polymorphic locus (rs2296957) in exon 6 and additional five intronic SNPs (rs3904060 and rs7867616, Intron 1; rs2296956 and rs189473964, Intron 3; rs150900763, 5' UTR) at the intron/exon boundaries. The presence of an individual genotype at these loci was not associated with therapy response and UCK1 gene expression.

Conclusions:
Our results show that absence of clinical response to azacitidine could be partly correlated with lower expression of UCK1. This is most likely causing a scarce azacitidine monophosphorylation by UCK1 in non-responders patients. The methylation status of the UCK1 gene promoter is not affecting gene expression. In addition, the genetic polymorphisms found in the UCK1 sequence do not associate with therapy response nor with gene expression. Further studies on larger series of MDS cases are needed to confirm these results which could help driving therapeutic decisions.

Disclosures:
No relevant conflicts of interest to declare.