Abstract
Introduction: Epigenetic alterations leading to the silencing of key tumor suppressor genes by promoter hypermethylation have been implicated in the pathogenesis of a number of malignancies, including MDS and AML. Currently, the prototypical DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (decitabine) has been studied in a number of diverse protocols as an anti-leukemic agent. However, the pattern of non-responsiveness to decitabine appears to be complex and multifactorial with some patients showing primary resistance whereas others develop resistance following initial responsiveness. To further understand the molecular mechanisms that define growth regulatory networks in pediatric AML, we have established and performed initial characterization using primary blasts that showed increased cell survival and proliferation in the presence of decitabine.
Methods: Bone marrow leukemic blasts from a relapsed pediatric AML patient, who received only conventional chemotherapy, were obtained following local REB approval and informed parental consent. Upon in vitro culture to identify effective therapeutic agents, enhanced cell survival was noted in wells containing decitabine (1uM) compared to untreated cells. This cell population was further expanded in higher concentrations of decitabine that tolerated concentrations higher than 10 uM. These cells were then clonally expanded, and the resulting cell line designated POETIC1, was screened in growth inhibition assays against a panel of 142 pharmaceutical pipe-line agents that target known growth regulatory pathways and signaling molecules. The original primary leukemic cells and normal lymphocytes were used as control. Gene expression analyses were carried out using humanHT-12 v4 Expression BeadChip whole-genome expression arrays, normalized and analyzed using the Illumina BeadStudio Software. The distribution and plasticity, and quantity of DNA methylation were studied using the Illumina Infinium Human Methylation BeadChip Assay.
Results: POETIC1 cells showed a differential drug sensitivity in approximately 20% of the agents tested. This includes enhanced susceptibility to agents that interfere with cell cycle regulation such as aurora kinase inhibitors, PLK, HDAC inhibitors and agents that targeted mTOR and proteasome activities. Transcriptome profiling revealed that 399 genes were down-regulated and 977 were up-regulated in the leukemia cells, compared to normal controls. POETIC 1 cells had significantly up-regulated DNA repair, cell cycle, oxidative phosphorylation and many other pro-survival pathways. Pathway analysis revealed that up-regulated genes belonged to cell cycle control and pro-survival signalling pathways including genes encoding for cyclins A and B, Cdc 7, Cdc 20 among others. They also had down-regulated genes relating to apoptosis, endocytosis and cell differentiation pathways. Global DNA methylome analysis revealed profound genome-wide deregulation of DNA methylation in POETIC 1 cells with a large number of genes were differentially methylated, including those involved in the control of cell cycle, oxidative phosphorylation, apoptosis and DNA repair pathways.
Discussion: Our findings indicate aberrant cell cycle and metabolic pathways in leukemia cells with primary resistance to decitabine. The POETIC1 cell line, provides a critical experimental tool to investigate the role of epigenetic alterations in leukemogenesis as well as the molecular and physiological mechanisms that define primary resistance to methyltransferase inhibitors and facilitate the identification of novel therapeutic agents for refractory disease in future clinical studies.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
