SENSITIZATION OF HEMATOPOIETIC STEM AND PROGENITOR CELLS TO TRIMETREXATE USING NUCLEOSIDE TRANSPORT INHIBITORS

Antifolates such as methotrexate (MTX) and trimetrexate (TMTX) are wid ely used in the treatment of cancer and nonmalignant disorders. Transi ent, yet sometimes severe myelosuppression is an important limitation to the use of these drugs. it has previously been shown that clonogeni c myeloid progenitors and colony-forming units-spleen are resistant to antifolates, suggesting that myelotoxicity occurs late in hematopoiet ic development. The goal of this study was to define the mechanisms by which primitive hematopoietic: cells resist the toxic effects of anti folate drugs. To test the hypothesis that myeloid progenitors may salv age extracellular nucleotide precursors to resist TMTX toxicity, a def ined liquid culture system was developed to measure TMTX toxicity in e xpanding progenitor populations. These in vitro experiments showed tha t both human and murine progenitors can resist TMTX toxicity by import ing thymidine and hypoxanthine from the serum. As predicted from these findings, several drugs that block thymidine transport sensitized pro genitors to TMTX in vitro, although to differing degrees. These nucleo side transport inhibitors were used to test whether progenitors and he matopoietic stem cells (HSCs) could be sensitized to TMTX in vivo. Tre atment of mice with TMTX and nitrobenzylmercaplopurineriboside phospha te (NBMPR-P), a potent transport inhibitor, caused significant depleti ons of clonogenic progenitors within the bone marrow (20-fold) and spl een (6-fold). Furthermore, NBMPR-P administration dramatically sensiti zed HSCs to TMTX, with dual-treated mice showing a greater than 90% re duction in bone marrow repopulating activity. These studies demonstrat e that both myeloid progenitor cells and HSCs resist TMTX by using nuc leotide salvage mechanisms and that these pathways can be pharmacologi cally blocked in vivo using nucleoside transport inhibitors. These res ults have important implications regarding the use of transport inhibi tors for cancer therapy and for using variants of dihydrofolate reduct ase for in vivo selection of genetically modified HSCs. (C) 1997 by Th e American Society of Hematology.