¹Institute of Human Genetics, Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN

^|[ast]|R. Scott McIvor has a financial interest in Discovery Genomics, Inc.

Abstract

Methotrexate (MTX) is an effective chemotherapeutic agent in the treatment of several proliferative tumors, most notably acute lymphocytic leukemia, Ewings sarcoma and osteosarcoma. However, methotrexate also has considerable toxicity for normal proliferative tissues, including hematopoietic cells and cells of the gastrointestinal tract. The toxicity of methotrexate thus limits its chemotherapeutic effectiveness. MTX acts by binding to dihydrofolate reductase (DHFR) as a competitive inhibitor of the enzyme. We are investigating the possibility of expressing drug-resistant forms of DHFR in hematopoietic cells as a means of protecting the recipient from the toxicity associated with MTX chemotherapy. We previously demonstrated substantial protection of recipient mice from lethal toxicity of MTX by transplantation of transgenic marrow expressing Arg22 or Tyr22 forms of drug-resistant DHFR. Here we report that the increased MTX dose tolerance afforded by transplantation with marrow expressing the tyr22 form of drug-resistant DHFR allows for improved chemotherapy of murine L1210 leukemia. In our experimental model, animals were administered a subcutaneous dose of 10e6 L1210 tumor cells. Three days later, the animals were given a sublethal (650 cGy) dose of Cs irradiation. Our preliminary studies demonstrated that this course of treatments left a consistent amount of residual disease in the animals. One day after irradiation, animals were transplanted with 107 bone marrow cells flushed either from normal animals or from Tyr22 DHFR transgenic animals, and on the following day MTX administration was initiated. Control mice administered PBS developed tumor at a median of 12 days after subcutaneous injection of L1210 cells. Administration of 4 mg/kg MTX resulted in a considerable delay in the emergence of tumor for groups given either normal BMT or DHFR transgenic BMT. However, animals transplanted with normal marrow suffered from MTX toxicity, evidenced by reduced hematocrit, while there was no anemia observed in animals transplanted with DHFR transgenic marrow. A second experiment was conducted in the same way except that MTX was administered at a higher dose of 10 mg/kg/day. In this experiment, all control animals transplanted with normal marrow succumbed to toxicity associated with drug administration, while animals transplanted with DHFR transgenic marrow were substantially protected from drug toxicity. Appearance of tumor was delayed to a median of 28 days in comparison to 12 days for PBS administered controls. We conclude that expression of drug-resistant DHFR in hematopoietic cells confers substantial resistance to MTX, which can be used for more effective chemotherapy against L1210 leukemia in mice. These results support the application of DHFR gene transfer as strategy for improved cancer chemotherapy using MTX and other antifolates.