Karin Skoglund

Explaining TPMT genotype/phenotype discrepancy by haplotyping of TPMT*3A and identification of a novel sequence variant, TPMT*23

Thiopurine methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism of thiopurine drugs. Owing to polymorphisms in the TPMT gene (TPMT*2–*22), the enzyme activity varies interindividually. Patients with reduced TPMT activity may develop adverse reactions when treated with standard doses of thiopurines. This work focuses on a TPMT genotype/phenotype discrepancy found in a patient during routine testing. The patient displayed very low TPMT enzyme activity and she was genotyped by pyrosequencing as being heterozygous for the 460G>A and 719A>G polymorphisms (TPMT*3A). Complete sequencing in combination with haplotyping of the TPMT gene revealed a novel sequence variant, 500C>G, on one allele and TPMT*3A on the other allele, giving rise to the novel genotype TPMT*3A/*23. When investigating the patients relatives, they too had the TPMT*3A/*23 genotype in combination with low enzyme activity. We conclude that this novel variant allele affects enzyme activity, as the individuals carrying it had almost undetectable TPMT activity.

Increased Sensitivity to Thiopurines in Methylthioadenosine Phosphorylase–Deleted Cancers

The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), are used in the treatment of leukemia. Incorporation of deoxythioguanosine nucleotides (dGs) into the DNA of thiopurine-treated cells causes cell death, but there is also evidence that thiopurine metabolites, particularly the 6-MP metabolite methylthioinosine monophosphate (MeTIMP), inhibit de novo purine synthesis (DNPS). The toxicity of DNPS inhibitors is influenced by methylthioadenosine phosphorylase (MTAP), a gene frequently deleted in cancers. Because the growth of MTAP-deleted tumor cells is dependent on DNPS or hypoxanthine salvage, we would predict such cells to show differential sensitivity to 6-MP and 6-TG. To test this hypothesis, sensitivity to 6-MP and 6-TG was compared in relation to MTAP status using cytotoxicity assays in two MTAP-deficient cell lines transfected to express MTAP: the T-cell acute lymphoblastic leukemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP−) transfected to express MTAP constitutively (A549-MTAP+). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intracellularly to MeTIMP, was markedly higher in both cell lines under MTAP− conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dGs incorporation is the main cause of cytotoxicity with 6-TG. These data suggest that thiopurines, particularly 6-MP, may be more effective in patients with deleted MTAP. Mol Cancer Ther; 10(3); 495–504. ©2011 AACR.

ABCB1 haplotypes do not influence transport or efficacy of tyrosine kinase inhibitors in vitro

Single-nucleotide polymorphisms (SNPs) in the gene coding for the efflux-transport protein ABCB1 (P-glycoprotein) are commonly inherited as haplotypes. ABCB1 SNPs and haplotypes have been suggested to influence the pharmacokinetics and therapeutic outcome of the tyrosine kinase inhibitor (TKI) imatinib, used for treatment of chronic myeloid leukemia (CML). However, no consensus has yet been reached with respect to the significance of variant ABCB1 in CML treatment. Functional studies of variant ABCB1 transport of imatinib as well as other TKIs might aid the interpretation of results from in vivo association studies, but are currently lacking. The aim of this study was to investigate the consequences of ABCB1 variant haplotypes for transport and efficacy of TKIs (imatinib, its major metabolite N-desmethyl imatinib [CGP74588], dasatinib, nilotinib, and bosutinib) in CML cells. Variant haplotypes – including the 61A>G, 1199G>A, 1236C>T, 1795G>A, 2677G>T/A, and 3435T>C SNPs – were constructed in ABCB1 complementary DNA and transduced to K562 cells using retroviral gene transfer. The ability of variant cells to express ABCB1 protein and protect against TKI cytotoxicity was investigated. It was found that dasatinib and the imatinib metabolite CGP74588 are effectively transported by ABCB1, while imatinib, nilotinib, and bosutinib are comparatively weaker ABCB1 substrates. None of the investigated haplotypes altered the protective effect of ABCB1 expression against TKI cytotoxicity. These findings imply that the ABCB1 haplotypes investigated here are not likely to influence TKI pharmacokinetics or therapeutic efficacy in vivo.

Single-nucleotide polymorphisms of ABCG2 increase the efficacy of tyrosine kinase inhibitors in the K562 chronic myeloid leukemia cell line.

Objective The tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukemia are substrates for the efflux transport protein ATP-binding cassette subfamily G member 2 (ABCG2). Variations in ABCG2 activity might influence pharmacokinetics and therapeutic outcome of TKIs. The role of ABCG2 single-nucleotide polymorphisms (SNPs) in TKI treatment is not clear and functional in-vitro studies are lacking. The aim of this study was to investigate the consequences of ABCG2 SNPs for transport and efficacy of TKIs [imatinib, N-desmethyl imatinib (CGP74588), dasatinib, nilotinib, and bosutinib]. Materials and methods ABCG2 SNPs 34G>A, 421C>A, 623T>C, 886G>C, 1574T>G, and 1582G>A were constructed from ABCG2 wild-type cDNA and transduced to K562 cells by retroviral gene transfer. Variant ABCG2 expression in cell membranes was evaluated and the effects of ABCG2 SNPs on transport and efficacy of TKIs were measured as the ability of ABCG2 variants to protect against TKI cytotoxicity. Results Wild-type ABCG2 had a protective effect against the cytotoxicity of all investigated compounds except bosutinib. It was found that ABCG2 expression provided better protection against CGP74588 than its parent compound, imatinib. ABCG2 421C>A, 623T>C, 886G>C, and 1574T>G reduced cell membrane expression of ABCG2 and the protective effect of ABCG2 against imatinib, CGP74588, dasatinib, and nilotinib cytotoxicity. Conclusion These findings show that the ABCG2 SNPs 421C>A, 623T>C, 886G>C, and 1574T>G increase the efficacy of investigated TKIs, indicating a reduced transport function that might influence TKI pharmacokinetics in vivo. Furthermore, the active imatinib metabolite CGP74588 is influenced by ABCG2 expression to a greater extent than the parent compound.

In vivo CYP3A activity and pharmacokinetics of imatinib in relation to therapeutic outcome in chronic myeloid leukemia patients.

Background: Cytochrome P450 3A (CYP3A) isoenzyme metabolic activity varies between individuals and is therefore a possible candidate of influence on the therapeutic outcome of the tyrosine kinase inhibitor imatinib in patients with chronic myeloid leukemia (CML). The aim of this study was to investigate the influence of CYP3A metabolic activity on the plasma concentration and outcome of imatinib in patients with CML. Methods: Forty-three patients with CML were phenotyped for CYP3A activity using quinine as a probe drug and evaluated for clinical response parameters. Plasma concentrations of imatinib and its main metabolite, CGP74588, were determined using liquid chromatography–mass spectrometry. Results: Patients with optimal response to imatinib after 12 months of therapy did not differ in CYP3A activity compared to nonoptimal responders (quinine metabolic ratio of 14.69 and 14.70, respectively; P = 0.966). Neither the imatinib plasma concentration nor the CGP74588/imatinib ratio was significantly associated with CYP3A activity. Conclusions: The CYP3A activity does not influence imatinib plasma concentrations or the therapeutic outcome. These results indicate that although imatinib is metabolized by CYP3A enzymes, this activity is not the rate-limiting step in imatinib metabolism and excretion. Future studies should focus on other pharmacokinetic processes so as to identify the major contributor to patient variability in imatinib plasma concentrations.

Imatinib reduces cholesterol uptake and matrix metalloproteinase activity in human THP-1 macrophages

BACKGROUND Imatinib mesylate (Glivec®, formerly STI-571) is a selective tyrosine kinase inhibitor used for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. However, there are reports suggesting that imatinib could be atheroprotective by lowering plasma low-density lipoprotein (LDL). AIM To investigate the potential inhibitory effect of imatinib on cholesterol uptake in human macrophages as well as its effect on matrix metalloproteinase (MMP) activity. METHODS AND RESULTS Uptake of fluorescence-labeled LDL was analyzed using flow cytometry. Macrophages treated with imatinib showed a 23.5%, 27%, and 15% decrease in uptake of native LDL (p<0.05), acetylated LDL (p<0.01), and copper-modified oxidized LDL (p<0.01), respectively. Gel-based zymography showed that secretion and activity of MMP-2 and MMP-9 were inhibited by imatinib. Using GeneChip Whole Transcript Expression array analysis, no obvious gene candidates involved in the mechanisms of cholesterol metabolism or MMP regulation were found to be affected by imatinib. Instead, we found that imatinib up-regulated microRNA 155 (miR155) by 43.8% and down-regulated ADAM metallopeptidase domain 28 (ADAM28) by 41.4%. Both genes could potentially play an atheroprotective role and would be interesting targets in future studies. CONCLUSION Our results indicate that imatinib causes post-translational inhibition with respect to cholesterol uptake and regulation of MMP-2 and MMP-9. More research is needed to further evaluate the role of imatinib in the regulation of other genes and processes.

Abstract 5596: Altered efficacy of tyrosine kinase inhibitors in chronic myeloid leukemia cells expressing wild type or polymorphic ABCG2

The tyrosine kinase inhibitor (TKI) imatinib is, despite the introduction of second generation TKI9s, the standard first-line therapy in chronic myeloid leukemia (CML). Early response to therapy is correlated to long-term therapeutic effect in CML; therefore it would be advantageous to identify predictive markers for failure on imatinib in order to enable a change of therapeutic strategy in an early stage of CML. Imatinib is metabolized by hepatic CYP3A and we have previously reported that high CYP3A activity is associated with a better therapeutic response, indicating a clinical significance of imatinib metabolites (Green et al. 2010). Furthermore, imatinib and the second generation TKI9s (dasatinib and nilotinib) are substrates for the efflux transporter ABCG2 which might be of importance for the elimination and resistance to TKIs. Several ABCG2 polymorphisms have been reported and some have been suggested to influence imatinib therapeutic outcome. In this study, we aimed to investigate the effects of expressing wild type (wt) or polymorphic ABCG2 on the in vitro resistance to imatinib, dasatinib, nilotinib, bosutinib (currently in clinical trials) and the most abundant CYP3A imatinib metabolite, CGP74588. The ABCG2 polymorphisms 34G>A, 421C>A, 623T>C, 886G>C, 1574T>G and 1582G>A were constructed from wt human ABCG2 cDNA. ABCG2 wt or variant cDNA was retrovirally infected to the CML cell line K562. Cell-surface ABCG2 expression was evaluated using FACS. In addition, K562/ABCG2/623T>C and K562/ABCG2/1574T>G were permeabilized to detect cytoplasmic ABCG2 protein. The influence of wt and variant ABCG2 on the cytotoxic effect of imatinib, CGP74588, dasatinib, nilotinib and bosutinib was assessed using MTT assays. Over-expression of ABCG2 wt in K562 cells conferred resistance to all TKI9s except bosutinib. The most striking effect was seen in cells treated with CGP74588 where ABCG2 wt expression induced a 7.8-fold increase in resistance compared to a moderate 2-fold increase in cells treated with imatinib. ABCG2 polymorphisms affected the cytotoxicity of imatinib, CGP74588, dasatinib and nilotinib in a similar way and did not seem to alter substrate specificity of the transporter. Polymorphisms 623T>C, 886G>C and 1574T>G completely sensitized cells to the level of non-ABCG2 expressing cells. In accordance, FACS analysis revealed that 623T>C and 1574T>G caused a failure of ABCG2 protein to localize to the cell surface but was yet detected in the cytoplasm. We conclude that CGP74588 cytotoxicity is more affected by ABCG2 expression than imatinib itself, indicating that ABCG2 activity might affect CGP74588 to a greater extent than imatinib in terms of plasma and target cell concentrations in vivo. ABCG2 with the polymorphisms 623T>C and 1574T>G does not incorporate into the cell membrane, affecting not only TKI transport but most likely all ABCG2 substrates. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5596. doi:1538-7445.AM2012-5596

Abstract 814: P-glycoprotein transport of the active imatinib metabolite, CGP74588, in chronic myeloid leukemia cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The tyrosine kinase inhibitor (TKI) imatinib is, despite the introduction of second generation TKIs, the standard first-line therapy in chronic myeloid leukemia (CML). Early response to therapy is correlated to long-term therapeutic effect in CML. To be able to switch to second generation TKIs in an early stage of CML, it would be advantageous to identify predictive markers for failure on imatinib. Imatinib is metabolized by hepatic CYP3A4 and CYP3A5, forming approximately 30 metabolites. The main metabolite, [CGP74588][1], is pharmacologically active with similar potency to that of imatinib. [CGP74588][1] is present in ∼20% of imatinib plasma concentrations but with a large inter-individual variation. We have previously reported that high CYP3A activity is associated with a better therapeutic outcome of imatinib, indicating a clinical significance of imatinib metabolites (Green et al. 2010). Furthermore, imatinib is a substrate for the efflux transporter P-glycoprotein which is the product of the ABCB1 gene. Several ABCB1 polymorphisms have been described and some have been shown to influence the therapeutic response to imatinib in CML patients. The aim of this study was to investigate the effects of ABCB1 over-expression on the in vitro resistance to imatinib and its CYP3A metabolite [CGP74588][1]. ABCB1 wild type human cDNA was infected to the CML cell line K562 using a retroviral system. Co-expression of ABCB1 and the reporter gene for enhanced yellow fluorescent protein (EYFP) was used for the assessment of ABCB1 expression by the analysis of EYFP in FACS. The influence of ABCB1 expression on the cytotoxic effects of imatinib and [CGP74588][1] was assessed using MTT assays. FACS analysis of EYFP confirmed an over-expression of ABCB1 in infected K562 cells with a mean fluorescence intensity of 23.5 compared to 0.55 in parental K562 cells. ABCB1-expressing cells (K562/ABCB1) were slightly, but not significantly, more resistance to imatinib than K562 cells (IC50 K562 = 0.41µM, K562/ABCB1 = 0.50µM). However, ABCB1 expression induced a 12-fold increase in resistance when cells were treated with the imatinib metabolite [CGP74588][1] (IC50 K562 = 0.72µM, K562/ABCB1 = 8.53µM, p = <0.000). Recent reports have shown that [CGP74588][1] accumulates in cell lines with acquired multi-drug resistance and high P-gp expression. Our studies on cells with ABCB1 expression as a single resistance mechanism proves that P-gp is indeed confer resistance to [CGP74588][1] in CML cells. Furthermore, [CGP74588][1] cytotoxicity is affected by ABCB1 expression to a greater extent than imatinib itself, indicating that ABCB1 activity could be important for [CGP74588][1] plasma and target cell concentrations in vivo and might contribute to the large intra-individual variation seen in patient plasma concentrations. Future studies will be needed to determine the clinical significance of P-gp activity and [CGP74588][1] pharmacokinetics in relation to TKI therapy of CML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 814. doi:1538-7445.AM2012-814 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=CGP74588&atom=%2Fcanres%2F72%2F8_Supplement%2F814.atom

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.