Molecularly evolved thymidylate synthase inhibits 5-fluorodeoxyuridine toxicity in human hematopoietic cells

Abstract

Thymidylate synthase (TS) inhibitors, such as 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FUdR), are amongst the most frequently used chemotherapeutic drugs available, although their efficacy is often limited by myelotoxicity. An emerging strategy for overcoming bone marrow toxicity involves ex vivo genetic transfer of drug resistance to autologous hematopoietic progenitor cells, followed by reimplantation of the transfected cells before chemotherapy. Here we establish that expression of mutant TS genes, selected from millions of engineered variants, renders human hematopoietic cells resistant to 5-FUdR, and identify the most efficacious variant for gene therapeutic rescue of drug-induced myelosuppression.

FIG. 1.

5-Fluorodeoxyuridine (5-FUdR) resistance conferred to human hematopoietic K562 cells by transduction of either wild-type thymidylate synthase (TS) or each of eight TS mutants. (a) Survival was quantified by clonogenic assay following culture for 3 days in the presence of increasing concentrations of 5-FUdR. Each point represents the mean ± SD of triplicate experiments. (b) The 50% lethal dose (LD₅₀), with 95% confidence limits, was determined by least-squares linear regression analysis of the survival curves. Color coding is the same in (a) and (b).

FIG. 2.

Amino acid substitutions in the eight mutants tested for 5-FUdR resistance in human cells are highlighted in the 1.9-Å crystal structure of dimeric human TS complexed with dUMP and the antifolate drug Raltitrexed. The closed conformation is shown. Residues 1–25 are disordered and do not appear in the model. The individual monomers are colored gray or green; for convenience, amino acid substitutions are indicated in only one of the monomers, although all substitutions are present in both. Comparison of LD₅₀ values among mutants (Fig. 1b) permitted the following differentiation of resistance-contributory versus provisionally neutral amino acid substitutions: Substitutions that either alone or together conferred enhanced 5-FUdR resistance (T51S, G52S) are colored red and orange. Substitutions present in mutants that conferred improved 5-FUdR resistance, but appear to be autonomous of this selected property (K82Q, K99E, N171S), are shown in yellow. Substitutions that alone or together did not confer greater 5-FUdR resistance than wild-type TS (D254N, T53S, Y258S, D116A, Y258F) are shown in blue.

FIG. 3.

Equal numbers of GFP expression-normalized wild-type and mutant TS-expressing K562 cells were sorted by FACS and cultured for 14 days (a) under standard cell culture conditions and (b) in medium supplemented with 500 nM 5-FUdR. Genomic DNA extracted from cells after 0, 3, 7, and 14 days of culture was used as template in PCRs employing primers that selectively amplified the retrovirally transduced TS genes. The resulting amplicons were cloned into the pCR 4-TOPO vector and transformed into DH5α Escherichia coli DH5α. The bacteria were plated and incubated overnight, and plasmids from the resultant colonies (n: 150 for each time point) were DNA sequenced with primers flanking the TS gene sequence.