Title: Dihydrofolate Reductase and Thymidylate Synthase Transgenes Resistant to Methotrexate Interact to Permit Novel Transgene Regulation Running Title: Methotrexate Resistance Permits Transgene Regulation

Methotrexate (MTX) is an antifolate which 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 a MTX-resistant DHFR DHFR L22F, F31S (DHFR) to rescue healthy cells. 5-fluorouracil (5-FU) resistant TYMS T51S, G52S (TYMS) is resistant to MTX and improves MTX resistance of DHFR in primary T cells. Here we find that a known mechanism of MTX-induced increase in DHFR expression persists with DHFR and cisexpressed transgenes. We also find that TYMS expression of cis-expressed transgenes is similarly decreased in an MTX-inducible manner. MTXinducible changes in DHFR and TYMS expression changes are lost when both genes are expressed together. In fact, expression of the DHFR and TYMS 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 interleukin – 12 (IL-12). Introduction: Anti-folate drugs have been in use for seven decades in the treatment of cancer. MTX is a commonly used agent from this class and inhibits several folate dependent enzymes including DHFR and TYMS. Inhibition of these proteins adversely affects the de novo synthesis of purine and thymidine nucleotides, which is vital for survival of rapidly replicating cells. MTX has proven valuable for treating rapidly replicating cancers such as leukemia, but the impact of MTX on healthy, replicating tissue leads to dose-limiting toxicities such as bone marrow suppression. 3 http://www.jbc.org/cgi/doi/10.1074/jbc.C115.671123 The latest version is at JBC Papers in Press. Published on August 4, 2015 as Manuscript C115.671123 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on July 6, 2017 hp://w w w .jb.org/ D ow nladed from