Protection of hematopoietic stem cells from pemetrexed toxicity by retroviral gene transfer with a mutant dihydrofolate reductase-mutant thymidylate synthase fusion gene.
Myelosuppression is one of the major side effects of most anticancer drugs. To confer myeloprotection, our laboratory generated drug-resistant mutants of select target human enzymes for gene transfer to the bone marrow. Mutants of two of these enzymes, dihydrofolate reductase (DHFR F/S) and thymidylate synthase (TS G52S), were previously shown to confer resistance to methotrexate and 5-FU, respectively, and recently a fusion cDNA of both mutant enzymes (DHFR F/S-TS G52S) was shown to confer dual resistance to both antimetabolites. In this study, we examined the sensitivity of the DHFR F/S-TS G52S fusion protein to the multitargeted antifolate, pemetrexed (LY231514, Alimta), which targets both DHFR and TS and is currently in phase III trials for the treatment of solid tumors and in combination with cisplatin has been shown to be an advance in the treatment of mesothelioma. The K(i) for the DHFR F/S portion of the purified fusion protein to pemetrexed was increased by greater than 9000-fold when compared to wtDHFR (8000 versus 0.86 nM), while the K(i) for the TS G52S portion of the fusion protein to pemetrexed was similar to that of wtTS (2.8 versus 3.1 nM). When the fusion gene was retrovirally transduced into NIH 3T3 fibroblasts, the IC(50) to pemetrexed was three- to four-fold higher than cells transduced with DHFR F/S or TS G52S alone (163 versus 53 and 45 nM, respectively). Similarly, expression of the DHFR F/S-TS G52S fusion gene in retrovirally transduced mouse marrow cells resulted in an increased survival of CFU-GM colonies when compared to cells transduced with either of the mutants alone. Co-expression of mutant DHFR and TS enzymes has additive effects in conferring resistance to pemetrexed-induced toxicity. This construct may be useful for conferring myeloprotection to patients receiving this drug.