Thymidylate Synthase Inhibition Assay Research Articles

Ex vivo resistance in childhood acute lymphoblastic leukemia: Correlations between BCRP, MRP1, MRP4 and MRP5 ABC transporter expression and intracellular methotrexate polyglutamate accumulation

Chemoresistance is an important factor in the treatment failure of childhood acute lymphoblastic leukemia (ALL). One underlying mechanism of chemoresistance involves (over)expression of ATP-dependent drug efflux transporters such as multidrug resistance protein 1–5 (MRP1–5) and breast cancer resistance protein (BCRP), which can extrude the important antileukemia drug methotrexate (MTX). Survival of childhood ALL critically depends on the leukemic blasts' capacity for intracellular retention of MTX and MTX-polyglutamates. This pilot study assessed whether expression of MRP1, MRP4, MRP5 and BCRP (real-time PCR) in primary childhood ALL blasts (n = 23) correlated with ex vivo resistance to MTX (assayed by in situ thymidylate synthase inhibition assay (TSIA)), ex vivo accumulation of (radioactive) MTX polyglutamates, and patient survival. Results show that high MRP4 expression is correlated with ex vivo MTX resistance assayed by TSIA (P = 0.01). Moreover, elevated MRP4 and BCRP expression correlated with lower accumulation of MTX-PGs (P = 0.004 and P = 0.03, respectively). Combined high expression of BCRP and MRP4 even further impacted reduced MTX-PG accumulation (P = 0.02). Overall survival was lower (P logrank = 0.04) in children with ALL cells which featured a relatively high expression of both BCRP and MRP4 transporters. These results underscore the impact of high drug efflux transporter expression, notably MRP4 and BCRP, in diminished MTX response in childhood ALL.

Abstract 4437: The relevance of aberrant FPGS splicing for ex vivo MTX resistance and clinical outcome in childhood acute lymphoblastic leukemia

Abstract Methotrexate (MTX) is one of the key components of current ALL treatment protocols. This antifolate inhibits de novo nucleotide biosynthesis, hindering DNA replication, hence leading to cell death. However, despite the overall good cure rates in ALL, drug resistance continues to hamper the efficacy of MTX, contributing to relapse of leukemia. Multiple molecular mechanisms underlying drug resistance have been characterized, however, their clinical relevance often remains unclear. We recently identified impaired FPGS splicing, in particular intron 8 partial retention (intron 8 PR), as a possible contributor to decreased FPGS activity in both MTX-resistant leukemia cell lines and primary ALL specimens. In the current study we explored the association between FPGS splicing alterations and MTX resistance as well as the clinical outcome of 91 pediatric ALL patients. With respect to MTX resistance, we determined the levels of MTX polyglutamates in leukemic cells, as well as FPGS activity along with the expression of several enzymes and transporters involved in (anti)folate metabolism and transport, including FPGS, FPGH, RFC, DHFR and TS. The sensitivity of leukemic cells to MTX was determined using the thymidylate synthase inhibition assay (TSIA). Moreover, the sensitivity to other chemotherapeutics as measured in the MTT assay was determined for these patient specimens. The levels of FPGS splicing alterations were semi-quantified using PCR and fragment analysis. We found that intron 8 PR plays an important role in MTX resistance in the studied patient cohort. In a subset of patients with relatively low levels of MTX polyglutamates, high levels of intron 8 PR were associated with inferior EFS (HR = 4.2, P = 0.006) and OS (HR = 5.6, P = 0.003). The relation between intron 8 PR and OS remained significant in multivariate analysis, which included known prognostic factors, including WBC and age. Interestingly, in this subgroup of patients, intron 8 PR correlated with higher MTX resistance as determined by the TSIA assay (p = 0.015) and lower accumulation of long chain MTX polyglutamates (p = 0.027). Moreover, high levels of intron 8 PR were associated with resistance to the glucocorticoids dexamethasone and prednisone. Remarkably, we found that a MTX-resistant FPGS-deficient subline of CCRF-CEM - CEM/FPGS-, harbors a high level of intron 8 PR and displays high level of dexamethasone cross-resistance (over 350-fold). Our findings suggest that intron 8 PR is not only relevant for MTX resistance but may also reflect a broader splicing defect affecting multiple genes, thereby resulting in multidrug resistance. These findings may have important implications for personalized treatment as well as circumvention of drug resistance in ALL patients. Financial support by Kika (Children cancer-free) Citation Format: Anna Wojtuszkiewicz, Yehuda G. Assaraf, Mirthe Hoekstra, Gerrit Jansen, Godefridus J. Peters, Edwin Sonneveld, Gertjan L. Kaspers, Jacqueline Cloos. The relevance of aberrant FPGS splicing for ex vivo MTX resistance and clinical outcome in childhood acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4437. doi:10.1158/1538-7445.AM2015-4437

Abstract 1624: Methotrexate resistance in relation to treatment outcome in childhood acute lymphoblastic leukemia

Abstract Methotrexate (MTX) is an essential component of contemporary ALL treatment regimens. This antifolate eradicates leukemic cells by disrupting de novo biosynthesis of nucleotides, leading to inhibition of DNA replication and consequent cell death. Over the past decades, MTX-containing chemotherapy has proven to be crucial in achieving curation in ALL. However, drug resistance continues to be a major obstacle to curative ALL treatment. A number of alterations in MTX metabolism, resulting in impaired accumulation of this antifolate in cancer cells, have been identified as determinants of MTX resistance. However, it remains unclear which of these molecular mechanisms have clinical relevance. Therefore, the current study aimed at determining the relation between MTX resistance and long-term clinical outcome of ALL. Towards this end, we collected the available clinical data of 235 childhood ALL patients, including clinical data with a follow-up time of 10 years. For these patients, samples obtained at the time of diagnosis were also characterized with respect to MTX resistance. This included determination of MTX polyglutamate levels in leukemic cells, mRNA expression of enzymes involved in (anti)folate metabolism and transport (i.e. FPGS, FPGH, DHFR, TS and RFC); MTX sensitivity was determined by the thymidylate synthase inhibition assay (TSIA) and in vitro FPGS activity assay was performed. High levels of long-chain MTX polyglutamates was strongly associated with favorable long-term event-free survival (EFS, p = 0.029) and overall survival (OS, p = 0.008) of ALL patients (10-year EFS 81% and OS 91%, compared to EFS 58% and OS 64% in patients with low levels of this active metabolite). Similar relations were observed for the total accumulation of MTX polyglutamates and FPGS activity, which were associated with overall survival (p = 0.018 and p = 0.039, respectively). In the multivariate Cox regression model, including clinical variables, the level of long chain MTX polyglutamates showed a trend towards an association with event-free survival (p = 0.073) but not with overall survival (p = 0.465). In addition, we assessed the association between MTX resistance-related variables and the treatment outcome in patients with different cytogenetic alterations. High MTX sensitivity was associated with hyperdiploid ALL (P<0.001), which was also associated with increased MTX accumulation (p = 0.03) and elevated RFC expression (p = 0.049). Moreover, cells characterized by TEL-AML1 fusion displayed elevated MTX resistance (p = 0.023) compared to samples devoid of this aberration, while MLL-rearrangements were associated with low accumulation of cellular MTX polyglutamates (p = 0.012). These findings emphasize the central role which MTX lays in ALL treatment thereby highlighting the necessity for further exploration of the molecular mechanisms underlying MTX resistance in ALL patients. Financial support by Kika (Children cancer-free) Citation Format: Anna Wojtuszkiewicz, Godefridus J. Peters, Nicole L. van Woerden, Boas Dubbelman, Gabrielle Escherich, Kjeld Schmiegelow, Edwin Sonneveld, Rob Pieters, Peter M. van de Ven, Gerrit Jansen, Yehuda G. Assaraf, Gertjan J L Kaspers, Jacqueline Cloos. Methotrexate resistance in relation to treatment outcome in childhood acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1624. doi:10.1158/1538-7445.AM2015-1624

Novel 1,3,4-oxadiazole thioether derivatives targeting thymidylate synthase as dual anticancer/antimicrobial agents

A series of novel 1,3,4-oxadiazole thioether derivatives (compounds 9–44) were designed and synthesized as potential inhibitors of thymidylate synthase (TS) and as anticancer agents. The in vitro anticancer activities of these compounds were evaluated against three cancer cell lines by the MTT method. Among all the designed compounds, compound 18 bearing a nitro substituent exhibited more potent in vitro anticancer activities with IC50 values of 0.7±0.2, 30.0±1.2, 18.3±1.4μM, respectively, which was superior to the positive control. In the further study, it was identified as the most potent inhibitor against two kinds of TS protein (for human TS and Escherichia coli TS, IC50 values: 0.62 and 0.47μM, respectively) in the TS inhibition assay in vitro and the most potent antibacterial agents with MIC (minimum inhibitory concentrations) of 1.56–3.13μg/mL against the tested four bacterial strains. Molecular docking and 3D-QSAR study supported that compound 18 can be selected as dual antitumor/antibacterial candidate in the future study.

Effect of polymorphisms in folate-related genes on in vitro methotrexate sensitivity in pediatric acute lymphoblastic leukemia

AbstractWe studied whether common polymorphisms in genes involved in folate metabolism affect methotrexate (MTX) sensitivity. Ex vivo MTX sensitivity of lymphoblasts obtained from pediatric patients with acute lymphoblastic leukemia (ALL; n = 157) was determined by the in situ thymidylate synthase inhibition assay after either continuous (21 hours; TSI50, cont) or short-term (3 hours; TSI50, short) MTX exposure. DNA was isolated from lymphoblasts obtained from cytospin slides. Polymorphisms in methylenetetrahydrofolate reductase (MTHFR 677C>T, MTHFR 1298A>C), methionine synthase (MTR 2756A>G), methionine synthase reductase (MTRR 66A>G), methylenetetrahydrofolate dehydrogenase (MTHFD1 1958G>A), serine hydroxymethyl transferase (SHMT1 1420C>T), thymidylate synthase (TS 2R3R), and the reduced folate carrier (RFC 80G>A) were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or real-time PCR. Patients with the MTHFR 1298AC variant or the MTRR 66 G-allele showed decreased in vitro MTX sensitivity measured under both test conditions. SHMT1 1420TT homozygotes only showed decreased MTX sensitivity in the TSI50, cont. In conclusion, polymorphisms in the folate-related genes MTHFR, MTRR, and SHMT1 are related to MTX resistance in pediatric patients with ALL. (Blood. 2005;106:717-720)

Effects and interaction of 7-hydroxy methotrexate and methotrexate in leukaemic cells ex vivo measured by the thymidylate synthase inhibition assay

In high dose therapy with methotrexate (MTX) the main metabolite 7-hydroxy-methotrexate (7-OH MTX) exceeds the plasma concentration of MTX achieving about tenfold higher levels. To investigate the interaction between 7-OH MTX and MTX ex vivo, the thymidylate synthase inhibition assay was used to quantify antifolate effects in patient blast samples, measuring the inhibition of the key enzyme thymidylate synthase (TS). In 18 leukemic samples (7 ALL, 11 AML) no dose-dependent TS inhibition was observed for 7-OH MTX. However, a statistically significant increase of TS inhibition (p<0.05) was observed for a 1:1 mixture of MTX and 7-OH MTX as compared to the effect of MTX alone. The half-maximal inhibitory concentrations in the short-exposure assay were 0.857 microM for MTX alone versus 0.088 microM for the 1:1 mixture with 7-OH MTX, respectively (p< or =0.05). This interaction was not observed with an excess of 7-OH MTX. Similar results were obtained in long exposure experiments. We conclude that there is a dose-dependent interaction between 7-OHMTX and MTX, despite the lack of TS inhibitory effects of the metabolite alone.

Effect and interaction of 7-hydroxy methotrexate and methotrexate in AML and ALL patient samples: measured by the thymidylate synthase inhibition assay

Effect and interaction of 7-hydroxy methotrexate and methotrexate in AML and ALL patient samples: measured by the thymidylate synthase inhibition assay

In vitro drug resistance and prognostic impact of p16INK4A/P15INK4B deletions in childhood T-cell acute lymphoblastic leukaemia.

p16 gene deletions are present in about 70% of primary paediatric T-cell acute lymphoblastic leukaemia (T-ALL) and 20% of common/precursor B-cell ALL cases. It is not clear what the impact of the frequent p16 deletions is within the subgroup of T-lineage ALL. We studied the relationship between p16/p19ARF deletions, using fluorescence in situ hybridization, and in vitro drug resistance and prognosis in childhood T-ALL at diagnosis. The cellular drug resistance was measured with the methyl thiazol tetrazoliumbromide assay using a panel of drugs and the thymidylate synthase inhibition assay for methotrexate. There was a complete overlap of individual LC50 values of p16 gene homozygously deleted and p16 germ-line cases for most of the nine classes of drugs tested. The only difference was for dexamethasone: the p16-deleted group was more sensitive than the germ-line p16 group (P = 0.030). The homozygously deleted p16 T-ALL patients (n = 34) treated with the modern multiagent chemotherapy schemes of the Dutch Childhood Leukaemia Study Group ALL-VII/-VIII or Co-operative ALL-92/-97 protocols have a significantly lower 5-year disease-free survival (DFS) than germ-line p16 T-ALL (n = 25) (65.1 +/- 9.1% vs. 95.5 +/- 4.4%, Plog rank = 0.021). Hence, this study identifies a subpopulation of primary childhood T-ALL that appears to have an extremely high DFS. However, the observed differences in outcome do not seem to be related to intrinsic resistance for the tested drugs.

Methotrexate resistance in relapsed childhood acute lymphoblastic leukaemia

Treatment failure in childhood acute lymphoblastic leukaemia (ALL) might be associated with methotrexate (MTX) resistance. Little is known about MTX resistance in relapsed ALL. In this study, we determined ex vivo MTX resistance in precursor-B ALL at relapse (rALL) and determined possible defects in MTX membrane transport and polyglutamylation. Using the in situ thymidylate synthase inhibition assay, 21 rALL samples were threefold more MTX resistant than 63 initial precursor-B ALL samples, both after short-term and after continuous MTX exposure (P < or = 0.01). [3H]-MTX membrane transport did not differ between eight rALL and 25 precursor-B ALL samples. Incubation for 24 h with 1 microM [3H]-MTX resulted in a trend towards a lower accumulation of MTX in 20 relapsed than in 83 initial samples of precursor-B ALL samples (906 vs. 1364 pmol/109 cells; P = 0.07). Accumulation of long-chain MTX polyglutamates (MTX-Glu4-6) did not differ between relapsed and newly diagnosed samples (746 and 889 pmol/109 cells; P = 0.1). Activities of the enzymes involved in polyglutamylation (folylpolyglutamate synthetase and folylpolyglutamate hydrolase) did not differ between rALL and untreated c/pre-B-ALL. This study demonstrates that leukaemic cells of children with relapsed precursor-B ALL are relatively MTX resistant, but that this MTX resistance is not associated with major impairments in MTX uptake or polyglutamylation.

Differential Methotrexate Resistance in Childhood T- Versus Common/PreB-Acute Lymphoblastic Leukemia Can Be Measured by an In Situ Thymidylate Synthase Inhibition Assay, But Not by the MTT Assay

Methotrexate (MTX) is not cytotoxic to patient-derived acute lymphoblastic leukemia (ALL) cells in total-cell-kill assays, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, putatively due to the rescue effects of hypoxanthine and thymidine released from dying cells. This was mimicked by a diminished methotrexate (MTX) cytotoxicity for the cell lines HL60 and U937 in the presence of hypoxanthine, thymidine, or lysed ALL cells. However, enzymatic depletion or inhibition of nucleoside membrane transport did not result in MTX dose-dependent cytotoxicity in patient samples. Alternatively, a thymidylate synthase inhibition assay (TSIA), based on inhibition of the TS-catalyzed conversion of 3H-dUMP to dTMP and 3H2O, correlated with the MTT assay for antifolate sensitivity in four human leukemia cell lines with different modes of MTX resistance. For 86 ALL patient samples, TSI50 values after 21 hours exposure to MTX were not different between T- and c/preB-ALL (P =.46). After 3 hours incubation with MTX followed by an 18-hour drug-free period, T-ALL samples were 3.4-fold more resistant to MTX compared with c/preB-ALL samples (P =.001) reflecting the clinical differences in MTX sensitivity. TSI50 values correlated with MTX accumulation (r = -.58, P <.001). In conclusion, the TSIA, but not the MTT assay, can measure dose-response curves for MTX in patient-derived ALL cells and showed relative MTX resistance in T-ALL compared with c/preB-ALL.

Differential Methotrexate Resistance in Childhood T- Versus Common/PreB-Acute Lymphoblastic Leukemia Can Be Measured by an In Situ Thymidylate Synthase Inhibition Assay, But Not by the MTT Assay

AbstractMethotrexate (MTX) is not cytotoxic to patient-derived acute lymphoblastic leukemia (ALL) cells in total-cell-kill assays, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, putatively due to the rescue effects of hypoxanthine and thymidine released from dying cells. This was mimicked by a diminished methotrexate (MTX) cytotoxicity for the cell lines HL60 and U937 in the presence of hypoxanthine, thymidine, or lysed ALL cells. However, enzymatic depletion or inhibition of nucleoside membrane transport did not result in MTX dose-dependent cytotoxicity in patient samples. Alternatively, a thymidylate synthase inhibition assay (TSIA), based on inhibition of the TS-catalyzed conversion of 3H-dUMP to dTMP and 3H2O, correlated with the MTT assay for antifolate sensitivity in four human leukemia cell lines with different modes of MTX resistance. For 86 ALL patient samples, TSI50 values after 21 hours exposure to MTX were not different between T- and c/preB-ALL (P = .46). After 3 hours incubation with MTX followed by an 18-hour drug-free period, T-ALL samples were 3.4-fold more resistant to MTX compared with c/preB-ALL samples (P = .001) reflecting the clinical differences in MTX sensitivity. TSI50 values correlated with MTX accumulation (r = −.58, P < .001). In conclusion, the TSIA, but not the MTT assay, can measure dose-response curves for MTX in patient-derived ALL cells and showed relative MTX resistance in T-ALL compared with c/preB-ALL.

Thymidylate Synthase Inhibition as a Predictor of Tumor Sensitivity of Fluorinated Pyrimidines

The thymidylate synthase (TS) content of tumor tissue was assayed and the levels of 5-fluorouracil (5-Fu) in serum and tumor tissue were determined in 21 patients with cervical cancer who were treated with UFT, a fluorinated pyrimidine. Subrenal capsule assay, a predictive tumor sensitivity test, was conducted concurrently on cervical tumor samples. TS inhibition and serum and tissue 5-Fu levels widely varied between both samples. Investigation of the relationship between the tumor growth inhibition rate determined by subrenal capsule assay and the above parameters (TS inhibition, serum and tissue 5-Fu levels) showed that TS inhibition and tumor growth inhibition were well correlated (r = 0.73). This suggests that a TS inhibition assay in tumor tissue can be a useful predictive test for tumor sensitivity of fluorinated pyrimidines.