Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2'-deoxycytidine in human leukemic KG1 cells.

Stéphane Vispé,Lucie Fournier,Natacha Novosad,Arthur Deroide,Fabrice Lestienne,Sophie Bréand,Paola B Arimondo,Emeline Davoine,Cécile Desjobert,Jörg Tost,Florence Busato,Didier Cussac,Luc De Vries,Joëlle Riond,Jérôme Besse

doi:10.18632/oncotarget.3317

Abstract

5-azacytidine and 5-aza-2'-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2'-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2'-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2'-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2'-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia.

Highlights

In recent years, epigenetic modifications in cancer have been largely investigated and have successfully led to the development of novel anticancer therapies aimed at reversing aberrant modification patterns, among which DNA methylation at cytosines in CpG dinucleotides is probably the best studied [1, 2]
Because KG1 www.impactjournals.com/oncotarget expresses all three DNMTs at high levels and it is known to possess several hypermethylated tumor suppressor genes (TSG) promoters, we chose it as in vitro human leukemia model to address the respective role of each DNMT in the maintenance of DNA methylation homeostasis
Since it has been shown in the human colon cancer cell line HCT116 that downregulation of DNMT1 by siRNA induced a demethylation of the promoter of CDKN2A [24], we tested in HCT116 the same DNMT1

Summary

Introduction

Epigenetic modifications in cancer have been largely investigated and have successfully led to the development of novel anticancer therapies aimed at reversing aberrant modification patterns, among which DNA methylation at cytosines in CpG dinucleotides is probably the best studied [1, 2]. At the same time promoters of many TSG become hypermethylated, associated with the silencing of the corresponding genes [3] Such deregulation has been used to identify and www.impactjournals.com/oncotarget develop DNA methylation inhibitors, among which two “epidrugs” have been approved to treat myelodysplasic syndromes (MDS), Acute Myeloid Leukemia (AML) and Chronic MyeloMonocytic Leukemia (CMML), namely 5-aza-2′-deoxycytidine (Dacogen®, hereafter named DAC) and 5-aza-2′cytidine (VidazaTM) [4,5,6,7]. These cytosine analogs target a family of proteins, called DNA Methyltransferases or DNMTs, responsible for the transfer of a methyl group onto a cytosine in the context of a CpG dinucleotide. These mechanisms are not selective for a specific DNMT, but rather involve all DNMTs acting on DNA

Methods

Results

Conclusion