Abstract
Drug resistance is a major problem in chemotherapy of leukemia. Several mechanisms of this phenomenon have been identified, but the underlying genomic changes are still poorly understood. Lack of drug sensitivity arises from a complex range of molecular events, which ultimately result in the blasts escaping death. Analysis of the gene expression profiles of cancer cells in correlation with in vitro cytotoxicity assay may define mechanisms of sensitivity and resistance to specific drugs. OBJECTIVE: To define and compare whole-genome responses to cytarabine (Ara-C), etoposide (VP16) and daunorubicin (DNR) in pediatric patients diagnosed with acute leukemias, and to explain in vitro chemoresistance phenotype of leukemic blasts.
In order to determine the ex vivo drug resistance profile, MTT cytotoxicity assay was performed on mononuclear cells obtained from 51 patients with ALL and 16 patients with AML. Gene expression profiles were prepared on the basis of cRNA hybridization to oligonucleotide arrays of the human genome (Affymetrix). Hierarchical clustering, assignment location and biological function were performed during the correlation analysis for identified probe sets. Verification of the relative expression level of genes (EGR1, GATA2, RUFY3, LDHA, DUSP2, BIN2, ICAM3, TTC28) was carried out by RT-qPCR in the study group and in an independent group of 53 patients.
Genetic expression profiles were identified, including those appropriate for ALL and AML: 181 and 106 genes for Ara-C, 274 and 314 for VP16, 146 and 495 for the DNR. For each of the drugs, a characteristic group of genes or processes that are responsible for the lack of sensitivity, were identified: (1) for Ara-C: overexpression of genes responsible for removing the drug from the cells, as well as changes in the nucleic acid metabolic process, especially transcription from RNA polymerase II promoter (eg. ZBTB16, FOSB, NFATC1, ZNF518B, PHF20L1 and RUFY); (2) for VP16: changes in expression level of genes involved in the regulation of mRNA transcription and DNA metabolism genes, including those controlling replication as well as those belonging to the double helix damage repair enzymes and genes participating in post-transcriptional mRNA splicing (eg. TOP2B, CSNK1E, BRIP1, ATR, MSH3 and MSH6); (3) for DNR: differentiated expression of factors involved in the replication and transcription processes, increased expression of kinases and intensification of the DNA repair processes (eg. ATF2, GATA2, TOX, RUNX3, MNDA, ST18, TFDP1, NFE2, SOX11 and PAX5). For each profile several common genes, such as: AGAP1, PRKCH, RAB31, BCL2A1, GCA, HLA-DRA, HLA-DPA1, IL8, RGS10, CEBPD, CLEC2, ANXA1, PLEK, S100A8, SLC, CXCL2, SOX, BTG, DEFA4 and TPD52, were identified. Pathway and functional gene ontology analysis showed that several features, independent of the initial type of leukemia cells and pattern of resistance include: overexpression of chemokines and hydrolases, increase in the expression of genes responsible for the maintenance of chromatin architecture, overexpression of anti-apoptotic genes, decrease in expression level of genes that promote apoptosis, decrease in gene expression of Wnt signaling pathway, down regulation of the expression of transcription factors, changes in the expression level of genes associated with the activation of B and T lymphocytes, differences in expression of genes responsible for the cell surface receptor linked signal transduction and intracellular signaling cascade.
This analysis showed that the mechanism of response to drugs is significantly genetically determined. Predictive sets of marker genes and functional groups for simultaneous assessment of the sensitivity to these 3 drugs, were identified. Many of these changes converge and ultimately lead to avoidance of apoptosis and further over-proliferation of cancer cells. This could also suggest that targeting these pathways as potential pharmacogenetics and therapeutic candidates may be useful for improving treatment outcomes in pediatric acute leukemias. This could also suggest that targeting these pathways as potential pharmacogenetics and therapeutic candidates may be useful for improving treatment outcomes.
This study was supported by Grant from the National Science Centre No. DEC-2011/03/D/NZ5/05749.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
