Anna Fyrberg

Decreased survival in normal karyotype AML with single-nucleotide polymorphisms in genes encoding the AraC metabolizing enzymes cytidine deaminase and 5'-nucleotidase.

De novo acute myeloid leukemia with normal karyotype (NK‐AML) comprises a large group of patients with no common cytogenetic alterations and with a large variation in treatment response. Single‐nucleotide polymorphisms (SNPs) in genes related to the metabolism of the nucleoside analogue AraC, the backbone in AML treatment, might affect drug sensitivity and treatment outcome. Therefore, SNPs may serve as prognostic biomarkers aiding clinicians in individualized treatment decisions, with the aim of improving patient outcomes. We analyzed polymorphisms in genes encoding cytidine deaminase (CDA 79A>C rs2072671 and −451C>T rs532545), 5′‐nucleotidase (cN‐II 7A>G rs10883841), and deoxycytidine kinase (DCK 3′UTR 948T>C rs4643786) in 205 de novo NK‐AML patients. In FLT3‐internal tandem duplication (ITD)‐positive patients, the CDA 79C/C and −451T/T genotypes were associated with shorter overall survival compared to other genotypes (5 vs. 24 months, P < 0.001 and 5 vs. 23 months, P = 0.015, respectively), and this was most pronounced in FLT3‐ITD‐positive/NPM1‐positive patients. We observed altered in vitro sensitivity to topoisomerase inhibitory drugs, but not to nucleoside analogues, and a decrease in global DNA methylation in cells carrying both CDA variant alleles. A shorter survival was also observed for the cN‐II variant allele, but only in FLT3‐ITD‐negative patients (25 vs. 31 months, P = 0.075). Our results indicate that polymorphisms in genes related to nucleoside analog drug metabolism may serve as prognostic markers in de novo NK‐AML. Am. J. Hematol. 88:1001–1006, 2013.

Optimization and evaluation of electroporation delivery of siRNA in the human leukemic CEM cell line

In order to study nucleoside analog activation in the CEM cell line, a transfection protocol had to be optimized in order to silence an enzyme involved in nucleoside analog activation. Hematopoetic cell lines can be difficult to transfect with traditional lipid-based transfection, so the electroporation technique was used. Field strength, pulse length, temperature, electroporation media, siRNA concentration, among other conditions were tested in order to obtain approximately 70–80% mRNA and enzyme activity downregulation of the cytosolic enzyme deoxycytidine kinase (dCK), necessary for nucleoside analog activation. Downregulation was assessed at mRNA and enzyme activity levels. After optimizing the protocol, a microarray analysis was performed in order to investigate whether the downregulation was specific. Additionally two genes were differentially expressed besides the downregulation of dCK. These were however of unknown function. The leakage of intracellular nucleotides was also addressed in the electroporated cells since it can affect the DNA repair mechansism and the efficiency of nucleoside analogs. Three of these pools were increased compared to untreated, unelectroporated cells. The siRNA transfected cells with reduced dCK expression and activity showed reduced sensitivity to several nucleoside analogs as expected. The multidrug resistance to other drugs, as seen in nucleoside analog-induced resistant cells, was not seen with this model.

Impact of ABCB1 single nucleotide polymorphisms 1236C>T and 2677G>T on overall survival in FLT3 wild‐type de novo AML patients with normal karyotype

Drug resistance is a clinically relevant problem in the treatment of acute myeloid leukaemia (AML). We have previously reported a relationship between single nucleotide polymorphisms (SNPs) of ABCB1, encoding the multi‐drug transporter P‐glycoprotein, and overall survival (OS) in normal karyotype (NK)‐AML. Here we extended this material, enabling subgroup analysis based on FLT3 and NPM1 status, to further elucidate the influence of ABCB1 SNPs. De novo NK‐AML patients (n = 201) were analysed for 1199G>A, 1236C>T, 2677G>T/A and 3435C>T, and correlations to outcome were investigated. FLT3 wild‐type 1236C/C patients have significantly shorter OS compared to patients carrying the variant allele; medians 20 vs. 49 months, respectively, P = 0·017. There was also an inferior outcome in FLT3 wild‐type 2677G/G patients compared to patients carrying the variant allele, median OS 20 vs. 35 months, respectively, P = 0·039. This was confirmed in Cox regression analysis. Our results indicate that ABCB1 1236C>T and 2677G>T may be used as prognostic markers to distinguish relatively high risk patients in the intermediate risk FLT3 wild‐type group, which may contribute to future individualizing of treatment strategies.

Cell Cycle Dependent Regulation of Deoxycytidine Kinase, Deoxyguanosine Kinase, and Cytosolic 5′-Nucleotidase I Activity in MOLT-4 Cells

Activation of nucleoside analogues is dependent on kinases and 5′-nucleotidases and the balance between the activity of these enzymes. The purpose of this study was to analyze deoxycytidine kinase, deoxyguanosine kinase, and 4 different 5′-nucleotidases during cell cycle progression in MOLT-4 cells. The activity of both kinases was cell cycle dependent and increased during proliferation while the activity of cytosolic 5′-nucleotidase I decreased. We could show that the kinase activity was higher than the total nucleotidase activity, which was unchanged or decreased during cell cycle progression. These data may be important in designing modern combination therapy with nucleoside analogues.

RNAi Depletion of Deoxycytidine and Deoxyguanosine Kinase in Human Leukemic CEM Cells

Resistance toward nucleoside analogues is often due to decreased activities of the activating enzymes deoxycytidine kinase (dCK) and/or deoxyguanosine kinase (dGK). With small interfering RNA (siRNA), dCK and dGK were downregulated by approximately 70% in CEM cells and tested against six nucleoside analogues using the methyl thiazol tetrazolium assay. SiRNA-transfected cells reduced in dCK activity were 3- to 6-fold less sensitive to CdA, AraC, and CAFdA. The sensitivity to AraG and FaraA was unchanged, while the sensitivity toward gemcitabine was significantly increased. dGK depletion in cells resulted in lower sensitivity to FaraA, dFdC, CAFdA, and AraG, but slightly higher sensitivity to CdA and AraC.

Nucleoside Analog Activity in Malignant Melanoma Cell Lines.

Mitochondrial deoxyguanosine kinase (dGK), is an enzyme responsible for activation of nucleoside analogs (NAs) to phosphorylated compounds which exert profound cytotoxicity, especially in hematological malignancies. Screening malignant melanoma cell lines against NAs revealed high sensitivity to several of them. This was believed to be due to the high levels of dGK expression in these cells. Downregulation of dGK in the melanoma cell line RaH5 using siRNA did not cause resistance to NAs as expected, but instead cells became more sensitive. This was probably partly due to the increased activity of another mitochondrial enzyme, thymidine kinase 2, seen in transfected cells.

Impact ofABCB1single nucleotide polymorphisms 1236C>T and 2677G>T on overall survival inFLT3wild-typede novoAML patients with normal karyotype

Drug resistance is a clinically relevant problem in the treatment of acute myeloid leukaemia (AML). We have previously reported a relationship between single nucleotide polymorphisms (SNPs) of ABCB1, encoding the multi‐drug transporter P‐glycoprotein, and overall survival (OS) in normal karyotype (NK)‐AML. Here we extended this material, enabling subgroup analysis based on FLT3 and NPM1 status, to further elucidate the influence of ABCB1 SNPs. De novo NK‐AML patients (n = 201) were analysed for 1199G>A, 1236C>T, 2677G>T/A and 3435C>T, and correlations to outcome were investigated. FLT3 wild‐type 1236C/C patients have significantly shorter OS compared to patients carrying the variant allele; medians 20 vs. 49 months, respectively, P = 0·017. There was also an inferior outcome in FLT3 wild‐type 2677G/G patients compared to patients carrying the variant allele, median OS 20 vs. 35 months, respectively, P = 0·039. This was confirmed in Cox regression analysis. Our results indicate that ABCB1 1236C>T and 2677G>T may be used as prognostic markers to distinguish relatively high risk patients in the intermediate risk FLT3 wild‐type group, which may contribute to future individualizing of treatment strategies.

Impact of ABCB1 single nucleotide polymorphisms 1236C greater than T and 2677G greater than T on overall survival in FLT3 wild-type de novo AML patients with normal karyotype

Drug resistance is a clinically relevant problem in the treatment of acute myeloid leukaemia (AML). We have previously reported a relationship between single nucleotide polymorphisms (SNPs) of ABCB1, encoding the multi‐drug transporter P‐glycoprotein, and overall survival (OS) in normal karyotype (NK)‐AML. Here we extended this material, enabling subgroup analysis based on FLT3 and NPM1 status, to further elucidate the influence of ABCB1 SNPs. De novo NK‐AML patients (n = 201) were analysed for 1199G>A, 1236C>T, 2677G>T/A and 3435C>T, and correlations to outcome were investigated. FLT3 wild‐type 1236C/C patients have significantly shorter OS compared to patients carrying the variant allele; medians 20 vs. 49 months, respectively, P = 0·017. There was also an inferior outcome in FLT3 wild‐type 2677G/G patients compared to patients carrying the variant allele, median OS 20 vs. 35 months, respectively, P = 0·039. This was confirmed in Cox regression analysis. Our results indicate that ABCB1 1236C>T and 2677G>T may be used as prognostic markers to distinguish relatively high risk patients in the intermediate risk FLT3 wild‐type group, which may contribute to future individualizing of treatment strategies.

Correlation between cytidine deaminase single nucleotide polymorphisms and in vitro drug sensitivity, DNA methylation and outcome in normal karyotype acute myelogenous leukemia

Correlation between cytidine deaminase single nucleotide polymorphisms and in vitro drug sensitivity, DNA methylation and outcome in normal karyotype acute myelogenous leukemia

A potential role of fetal hemoglobin in the development of multidrug resistance

Our previous data from a human leukemic cell line made resistant to the nucleoside analog (NA) 9-β-D-arabinofuranosylguanine (AraG) revealed a massive upregulation of fetal hemoglobin (HbF) genes and the ABCB1 gene coding for the multidrug resistance P-glycoprotein (P-gp). The expression of these genes is regulated through the same mechanisms, with activation of the p38-MAPK pathway and inhibition of methylation making transcription factors more accessible to activate these genes. We could show that AraG, as well as other NAs, and P-gp substrates could induce global DNA demethylation and induction of Hbγ and P-gp both at the mRNA and protein expression level. We speculate that the expression of HbF prior to drug exposure or in drug-resistant cell lines is a strategy of the cancer to gain more oxygen, and thereby survival benefits. We also believe that P-gp may be induced in order to excrete Hb degradation products from the cells that would otherwise be toxic. By using Hbγ siRNA and pharmacological inhibitors of HbF production we here present a possible relationship between HbF induction and multi-drug resistance in a human leukemia cell line model.