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Cancer research: from folate antagonism to molecular targets

https://doi.org/10.1016/j.beha.2009.09.004Get rights and content

The antifolates aminopterin and methotrexate have two firsts in the treatment of malignancy. Aminopterin was the first drug reported to cause remissions in children with acute lymphocytic leukaemia, and methotrexate (MTX), the antifolate that has supplemented aminopterin in the clinic, was the first drug that was shown to be curative for patients with a solid tumour, choriocarcinoma. More than 50 years after its introduction in the clinic, MTX is still being used and studied. The role of dihydrofolate reductase (DHFR), the principal target of aminopterin, has been studied extensively, and DHFR gene amplification and mutations have been implicated in drug resistance. Recent research focusses on studies of the translational regulation of DHFR and transfer of mutant DHFR and other drug resistance genes by viral vectors to protect haematopoietic cells. Based upon the detailed understanding of the mechanism of action of antifolates, both as inhibitors of DHFR and thymidylate syntase (TS), new agents have been developed that show effectiveness in the treatment of human malignancies. MTX remains a potent and widely used agent.

Section snippets

Dihydrofolate reductase and methotrexate

During the second and third year of my haematology fellowship with Clement A. Finch at the University of Washington, I was fortunate to work with Frank Huennekens, an assistant professor of biochemistry, who had initiated a programme to work out the enzymology of folate enzymes. The year before I joined the lab, in 1958, 10 years after MTX was introduced in the clinic, the mechanism of the action of MTX as a potent inhibitor of dihydrofolate reductase (DHFR) was established in a landmark paper

Methotrexate and beyond

At Yale, my colleagues and I initiated studies of high-dose MTX and leucovorin rescue for the treatment of head and neck cancer and diffuse large cell lymphoma [4], [5]. New anti-tumour agents, including trimetrexate and carboxypetidase G2(glucarpidase), were discovered [6]. Compared with methotrexate, trimetrexate is a quinazoline and lacks the terminal glutamate moiety (Fig. 1). Trimetrexate also has the property of being able to enter cells without the folate carriers needed by methotrexate.

Methotrexate resistance

While on sabbatical with Robert Schimke at the Stanford University, we discovered DHFR gene amplification as a mechanism of methotrexate resistance [15]. This discovery was remarkable because, at that time, it was believed that DNA was stable, and the concept of somatic cells generating additional gene copies was novel. Subsequently, it has been shown that resistance to other tight binding enzyme inhibitors could also be attributed to gene amplification.

At the Memorial Sloan-Kettering Cancer

Drug resistance genes

Mutations and overexpressed target proteins, due to gene amplification or other mechanisms, can confer resistance to drugs. Using this knowledge, others and we have conducted studies to determine the feasibility of protecting normal haematopoietic precursors from chemotherapy by transfer of drug resistance genes via retroviral constructs into marrow stem cells (reviewed by Budak-Alpdogan et al [17].). The rationale behind this approach is that tumours develop resistance to chemotherapy while

Translational regulation of DHFR

Our early studies showed that DHFR levels (bound tightly to MTX) increase within hours in both normal leucocytes and acute leukaemia cells after treatment with MTX. As MTX slowly dissociates from the enzyme, cells that survive treatment synthesise tetrahydrofolate and continue to proliferate. Our work[24] and the studies of Chu et al [25]. have shown that DHFR translation is inhibited by binding of the DHFR protein to its cognate mRNA. Translation inhibition due to DHFR enzyme is relieved by

Conclusions

Probably more is known about the mechanism of action of antifolates than any other class of drugs. Folate antagonists have been useful for the treatment of many diseases, ranging from bacterial infections, malaria, autoimmune disease and cancer. Knowledge of their mechanism of action continues to grow, giving rise to new antifolates and new uses of these drugs. To paraphrase the late George Hitchings, “understanding the mechanism of action of a drug can open many doors.”

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