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Methotrexate resistance in an in vivo mouse tumor due to a non-active-site dihydrofolate reductase mutation
A P Dicker1, M C Waltham, M Volkenandt
1Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Abstract:
A series of methotrexate (MTX)-resistant L1210 leukemia murine ascites tumors were developed in vivo and analyzed for drug resistance. Three of 20 tumors studied expressed an altered dihydrofolate reductase (DHFR) and each was identical, having a C to T base transition at nucleotide 46 in the DHFR gene as demonstrated by PCR and direct sequencing. This transition results in a Gly to Trp substitution at amino acid 15 of the enzyme. Purified altered enzyme displays significantly lower binding affinity for the antifolates MTX, trimetrexate, edatrexate, and trimethoprim with respective Ki values 165-, 76-, 30-, and 28-fold higher than values obtained for enzyme isolated from parental tumor (wild-type enzyme). Substrate (dihydrofolate) and cofactor (NADPH) binding is also diminished for the mutant enzyme, although to a lesser extent (17.3- and 3.6-fold higher Km, respectively). Gly-15 is highly conserved for all vertebrate species of DHFR but has no known interaction(s), either directly or indirectly, with bound cofactor, substrate, or inhibitor. Protein molecular modeling reveals that the affected residue is 9-12 A away from the enzyme active site and located in a region analogous to the mobile Met-20 loop domain characterized for Escherichia coli DHFR.
Insights
Methotrexate resistance in leukemia tumors is linked to a specific mutation in dihydrofolate reductase (DHFR). This genetic alteration significantly reduces the enzyme's drug-binding ability, impacting cancer treatment strategies.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Methotrexate (MTX) is a key antifolate drug used in chemotherapy.
- Drug resistance is a major challenge in treating cancers like L1210 leukemia.
- Dihydrofolate reductase (DHFR) is the molecular target of MTX.
Purpose of the Study:
- To investigate the molecular mechanisms of MTX resistance in L1210 leukemia.
- To identify genetic and enzymatic alterations associated with drug resistance.
Main Methods:
- Development of MTX-resistant L1210 murine ascites tumors in vivo.
- Polymerase Chain Reaction (PCR) and direct sequencing to analyze the DHFR gene.
- Enzyme kinetics studies to assess binding affinities for antifolates, substrate, and cofactor.
- Protein molecular modeling to visualize the structural impact of mutations.
Main Results:
- Three out of 20 MTX-resistant tumors exhibited an identical mutation in the DHFR gene (C to T at nucleotide 46).
- This mutation leads to a Glycine to Tryptophan substitution at amino acid 15 (Gly15Trp).
- The mutant DHFR enzyme showed significantly reduced binding affinity for MTX and other antifolates (165-fold for MTX), as well as diminished substrate and cofactor binding.
Conclusions:
- A specific mutation in DHFR (Gly15Trp) is responsible for MTX resistance in L1210 leukemia.
- This mutation affects antifolate binding despite Gly-15 not being in the active site, suggesting allosteric effects.
- Understanding this resistance mechanism can inform the development of new therapeutic strategies against resistant cancers.