Cellular ATP Depletion by LY309887 as a Predictor of Growth Inhibition in Human Tumor Cell Lines 1 Xiaohong Lu, Julie Errington, Victor J. Chen, Nicola J. Curtin, Alan V. Boddy, and David R. Newell 2 Cancer Research Unit, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United Kingdom [X. L., J. E., N. J. C., A. V. B., D. R. N], and Lilly Research Laboratories, Indianapolis, Indiana 46285 [V. J. C] ABSTRACT The antifolate LY309887 is a specific glycinamide ribo- nucleotide formyltransferase inhibitor that blocks de novo purine synthesis and produces a depletion of purine nucle- otides. The activity of LY309887 in six human tumor cell lines has been examined by growth inhibition and clonogenic assay after continuous exposure for three cell doubling times and by ATP depletion at 24 h. Three cell lines (CCRF-CEM, MCF7, and GC3) were sensitive to LY309887-induced growth inhibition (IC 50 : 5.6 – 8.1 nM), whereas the other cell lines (COR-L23, T-47D, and A549) were comparatively re- sistant (IC 50 : 36–55 nM). Sensitivity to LY309887 cytotoxic- ity was consistent with sensitivity to growth inhibition in four of five cell lines tested (MCF7/GC3: 0.01% survival and COR-L23/T-47D: 1–5% survival at 100 nM LY309887). LY309887-induced ATP depletion was measured by lucif- erase-based ATP assay and confirmed by high performance liquid chromatography measurements. There was a linear relationship between ATP depletion and growth inhibition when data were analyzed for all six cell lines (r 2  0.93; P < 0.0001). Depletion of 24-h cellular ATP concentrations to <1 mM was associated with both cell growth inhibition and cytotoxicity in all cell lines studied. In conclusion, cellular ATP depletion induced by LY309887 can be used to predict growth inhibition and cytotoxicity in human tumor cells. INTRODUCTION The folate-requiring enzymes that catalyze the de novo synthesis of nucleotides for DNA replication have been studied as targets for cancer therapy for many years. GARFT 3 (EC 2.1.2.1) is the first folate-dependent enzyme in the purine nu- cleotide synthetic pathway and uses N 10 -formyl-tetrahydrofolic acid as a cosubstrate. Inhibition of GARFT results in depletion of cellular purine nucleotide pools, which are necessary for cellular energy requiring processes and for the synthesis of DNA and RNA. Lometrexol (6R-5,10-dideaza-5,6,7,8-tetrahy- drofolic acid, 6R-DDATHF) is a specific GARFT inhibitor, which has potent antitumor activity against a number of murine tumors and human tumor xenografts (1). Consistent with the proposed mechanism of action of lometrexol, the ATP and GTP content of L1210 cells was decreased by at least 70% following treatment with lometrexol at concentrations causing growth inhibition (10 –100 nM; Ref. 2). LY309887 (6R-2',5'-thienyl-5,10-dideazatetrahydrofolic acid) is a thiophene analogue of lometrexol, and it is a repre- sentative second generation GARFT inhibitor, which is a more potent cytotoxic agent in vitro with greater antitumor activity than lometrexol in vivo (1, 3). Measurement of nucleotide pools following LY309887 treatment demonstrated a marked decrease in (d)ATP and (d)GTP levels within 6 h in CCRF-CEM cells (4). Furthermore, the growth inhibitory activity of both lome- trexol and LY309887 was reversed by HPX, but not by thymi- dine, reflecting the selective inhibition of de novo purine syn- thesis by both drugs (1, 2). During the clinical evaluation of lometrexol and LY309887, antitumor activity was reported in patients with malignant histiocytoma, breast, non-small cell lung, ovarian, and head and neck cancer (5–9). In initial clinical trials, lometrexol caused cumulative antiproliferative toxicities in the form of myelosuppression and gastrointestinal damage. Subsequently, it was shown that coadministration of folic acid ameliorates the cumulative antiproliferative toxicities of lome- trexol, thereby allowing substantial dose escalation (6). All clinical studies with LY309887, therefore, have included folic acid supplementation to reduce the cumulative aspects of the toxicity profile. Using structure-based drug design based on the X-ray structure of GARFT, the antipurine antifolate AG2034 (4-[2-(2- amino-4-oxo-4,6,7,8,-tetrahydro-[ 3 H]pyrimidino[5,4 – 6][1,4]- thiazin-6-yl)-(S)-ethyl]-2,5-thienoyl- L-glutamic acid) has recently been developed. AG2034 is also a thiophene analogue of lometrexol and has potent in vivo antitumor activity against a variety of murine and human tumor xenografts (10). As classical antifolates, all of the GARFT inhibitors developed to date are subject to the same biochemical determinants of activity as natural folates, notably membrane transport and intracellular polyglutamation. Received 8/2/99; revised 10/12/99; accepted 10/13/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Eli Lilly Co. and the Cancer Research Campaign (United Kingdom). 2 To whom requests for reprints should be addressed, at the Cancer Research Unit, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United Kingdom. Phone: 44-191-222- 5712; Fax: 44-191-222-7556; E-mail: herbie.newell@newcastle.ac.uk. 3 The abbreviations used are: GARFT, glycinamide ribonucleotide formyltransferase; HPX, hypoxanthine; HPLC, high performance liquid chromatography; TS, thymidylate synthase. 271 Vol. 5, 271–277, January 1, 2000 Clinical Cancer Research