EDITORIALS

Enhancing Cytotoxic Sensitivity of Tumor Cells to Antifolates: Another Opportunity for Gene Therapy?

F. M. Sirotnak

Correspondence to: F. M. Sirotnak, Ph.D., Laboratory of Molecular Therapeutics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021 (e-mail: sirotnaf{at}mskcc.org).

Effective cytotoxic action of classical folate analogues against tumor cells (1,2) relies not only on their potent inhibition of the primary intracellular target—analogues have been tailored to inhibit dihydrofolate reductase, thymidylate synthase, and glycinamide ribonucleotide formyl transferase—but also on their efficiently mediated internalization and subsequent conversion to poly-{gamma}-glutamates. As with natural folates, internalization of folate analogues by tumor cells [reviewed in (2)] is mediated by a plasma membrane transporter encoded by the RFC-1 gene. The enzymatic conversion of the internalized folate analogue to poly-{gamma}-glutamates occurs by amide bond formation at the {gamma}-carboxyl group of the resident glutamyl moiety of the folate analogue with the amino group of a second glutamate (1,3). A series of these reactions, mediated by folylpolyglutamate synthetase (FPGS) (4-6), results in the formation of poly-{gamma}-glutamates incorporating one to several additional glutamates. Poly-{gamma}-glutamates of folate analogues are retained more readily in tumor cells as well as in other mammalian tissues than is the parent analogue (7-9); as a result, poly-{gamma}-glutamates produce a more prolonged inhibition of the target enzyme and suppression of macromolecular biosynthesis. Also, poly-{gamma}-glutamates of 4-amino folate analogues such as methotrexate are often more effective as inhibitors of folate-dependent enzymes that are involved in macromolecular biosynthesis (1,10,11)—thus potentially targeting these enzyme directly. Net intracellular accumulation of poly-{gamma}-glutamate derivatives of folate analogues also reflects the action of folylpolyglutamate hydrolase (FPGH; also known as {gamma}-glutamyl hydrolase) (12), which mediates the hydrolysis of these compounds and their turnover following internalization into lysosomes (13).

The importance of poly-{gamma}-glutamate formation to the cytotoxicity of folate analogues was originally inferred from research (9) associating the degree of cytotoxicity with the extent of poly-{gamma}-glutamylation of structurally different analogues by various tumor cells. Further support for this notion was derived in other studies (14-16) showing that tumor cell variants with acquired resistance to folate analogues often had low levels of FPGS activity or gene expression compared with drug-naive parental cells or had high levels of FPGH (17). Additional evidence has accumulated subsequently (18) and strongly suggests that the natural refractoriness of some human cancers in a clinical setting to methotrexate may be due, in large measure, to its accumulation as poly-{gamma}-glutamates at only low levels.

Taking advantage of this extensive background of information on folate analogues, in this issue of the Journal, Aghi et al. (19) present studies that sought to enhance by gene transfer the cytotoxic sensitivity to folate analogues of rat gliosarcoma cells and other animal and human tumors originating in the central nervous system. The approach that they used was to transfect these cells with a plasmid or viral construct encoding human FPGS in the form of a complementary DNA (cDNA) prior to exposing them to a 4-aminofolate, dihydrofolate reductase inhibitor. This approach was somewhat similar to attempts by these authors (20) and others (21,22) to sensitize tumor cells to the cytotoxic action of pyrimidine and purine nucleosides by transfecting these cells with a viral thymidine kinase or deoxycytidine kinase. However, in contrast to these approaches, which attempt to enhance cytotoxicity by increased conversion to the proximate antimetabolite (pyrimidine or purine nucleotide), the elevated FPGS expression in the transfected tumors resulted in higher levels of intracellular accumulation and retention of the folate analogue as poly-{gamma}-glutamate metabolites.

What do the authors' findings reveal? Other investigators (23) have already shown that vector-mediated transfection of FPGS cDNA can restore FPGS activity, reverse auxotrophy, and reintroduce cytotoxic sensitivity into variant hamster cells that express no endogenous FPGS activity. These results provided the most direct proof of the importance of FPGS as a determinant of cytotoxicity of classical folate analogues. What Aghi et al. (19) have done is to introduce the notion of vector-mediated gene therapy and to demonstrate in this context that elevation of FPGS activity beyond the endogenous level characteristic of most target tumor cells will augment their cytotoxic sensitivity. Moreover, the authors provide further support for this notion by also showing that the impact of transfection was greater in the case of edatrexate (24), a more efficient substrate for FPGS, but nonexistent in the case of PT523 (25), an analogue that is not a substrate for FPGS. Of interest, the authors go on to provide evidence for a bystander effect (26), seemingly related to the gradual and prolonged release of folate analogues into the extracellular environment, that was more pronounced in the case of edatrexate. They also present some results suggesting that the impact of FPGS gene transfer on cytotoxic sensitivity is also manifested as an increase in therapeutic responsiveness to these folate analogues in a corresponding in vivo model of gliosarcoma.

What is the significance of the studies by Aghi et al.? Their data that show enhanced cytotoxic sensitivity of tumor cells to folate analogues following FPGS gene transfer provide—within a limited context—a proof-of-principle for the validity of an antifolate-based approach to gene therapy. Although these results are provocative and this approach may ultimately have valuable application in the case of human cancer, the results must be considered preliminary, as the authors have stated. Substantially more study will be required before the true merit of this approach will be apparent. The authors' experiments were carried out primarily with stably transfected, clonal cell lines, a far cry from the unselected delivery of exogenous genes to target tumors that will be required in clinically relevant model systems in vivo, let alone in human tumors in situ. The authors have taken their studies one step further toward clinical relevance by providing evidence for the impact of unselected transfer of the FPGS gene on cytotoxic sensitivity of a target tumor to edatrexate. In addition, they provide some documentation for a favorable bystander effect in the case of both selected and unselected gene transfer. This is an important prerequisite for success under real-world conditions, where the transfer of genes to cells within a tumor is substantially less than 100%. Given the usual strategic and tactical limitations associated with gene transfer, it is unclear at this time to what extent the authors' approach will compare favorably with other approaches that have a different pharmacologic focus and end point. The large body of knowledge available pertaining to the cellular pharmacology of folate analogues [reviewed in (1,2)] makes the authors' approach fundamentally appealing and provides a substantial conceptual advantage. However, one highly probable limitation that may present a major obstacle to widespread clinical application of this approach comes to mind: Many tumors that respond poorly to folate analogues do so (27) because of the less than optimal level of internalization of these agents by the RFC-1-encoded folate transporter. Since permeation of folate analogues into the cell by action of this transporter controls the amount of substrate available for FPGS, increased activity of this enzyme following FPGS gene transfer may have little impact on the ability of the tumor to generate poly-{gamma}-glutamates. Furthermore, there is also the question of tumor-specific delivery. How this will be achieved will be important to the eventual success of this therapy, since normal, proliferative cells in the gastrointestinal tract and bone marrow are also capable, to their detriment, of forming poly-{gamma}-glutamates from these analogues [reviewed in (1)]. Despite these potential limitations, an extension of the studies of Aghi et al. appears warranted and, with time and reasonable effort, should give us some indication of the intrinsic merit of this approach and its possible clinical applications.

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