Abstract
Methotrexate (MTX) is an anti-folate that inhibits de novo purine and thymidine nucleotide synthesis. MTX induces death in rapidly replicating cells and is used in the treatment of multiple cancers. MTX inhibits thymidine synthesis by targeting dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS). The use of MTX to treat cancer also causes bone marrow suppression and inhibits the immune system. This has led to the development of an MTX-resistant DHFR, DHFR L22F, F31S (DHFR(FS)), to rescue healthy cells. 5-Fluorouracil-resistant TYMS T51S, G52S (TYMS(SS)) is resistant to MTX and improves MTX resistance of DHFR(FS) in primary T cells. Here we find that a known mechanism of MTX-induced increase in DHFR expression persists with DHFR(FS) and cis-expressed transgenes. We also find that TYMS(SS) expression of cis-expressed transgenes is similarly decreased in an MTX-inducible manner. MTX-inducible changes in DHFR(FS) and TYMS(SS) expression changes are lost when both genes are expressed together. In fact, expression of the DHFR(FS) and TYMS(SS) cis-expressed transgenes becomes correlated. These findings provide the basis for an unrecognized post-transcriptional mechanism that functionally links expression of DHFR and TYMS. These findings were made in genetically modified primary human T cells and have a clear potential for use in clinical applications where gene expression needs to be regulated by drug or maintained at a specific expression level. We demonstrate a potential application of this system in the controlled expression of systemically toxic cytokine IL-12.
Highlights
Methotrexate targets dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS) to prevent cancer growth, and increases DHFR expression when applied
We attempted to recapitulate an increase in the expression of MTX-resistant DHFRFS after the administration of MTX within human T cells, which Skacel et al [18] has shown in a hamster cell line
As a control for our studies, DHFRFS transgene was codon-optimized to remove any of the mRNA sequence or structure that contributed to mRNA binding [16]
Summary
Methotrexate targets dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS) to prevent cancer growth, and increases DHFR expression when applied. In performing these experiments in primary human T cells, we identified that the application of MTX decreases TYMSSS expression in an MTX-dependent manner, likely due to TYMS binding its own mRNA to repress translation [19]. We present methods to increase, decrease, or link the expression of transgenes based on the use of mutant DHFRFS and TYMSSS in a clinically relevant context This system will permit new forms of post-translational regulation to be utilized for improved control of gene therapy vectors
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