a
`3
`
`g
`
`Cancer Chemotherapy
`and Biological Response
`Modifiers Annual 18
`
`Edited by
`
`H.M. Pinedo
`The Free University
`Amsterdam. The Netherlands
`
`D.L. Longo
`National Institute on Aging
`Baltimore, MD, U.S.A.
`
`B.A. Chabner
`Massachusetts General Hospital
`Boston, MA, U.S.A.
`
`1999
`
`L A
`.... .'
`
`Elsevier
`Amsterdam — Lausarme — New York —- Oxford - Shannon — Tokyo
`
`f_
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`Lil
`Sandoz V. Lilly IPR2
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`x. 2076
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`

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`First edition 1999
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`

`
`Cancer Chemotherapy and Biological Response Modifiers Annual 18
`
`
`
`
`
`
`
`HM. Pinedo, D.L. Longo and B.A. Chabncr. editors
`
`
`
`
`
`
`
`©1999 Elsevier Science B.V. All rights reserved.
`
`
`
`
`
`
`
`CHAPTER 1
`
`
`Antimetabolites
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`J.L. Grem, C.H. Takimoto, P. Multani, E. Chu, D. Ryan, B.A. Chabner, C.J. Allegra
`
`
`and P.G. Johnston
`
`1.
`
`Introduction
`
`
`
`
`2.1. Mechanism ofaction
`
`
`
`
`
`
`
`
`Ongoing basic research continues to focus on the
`
`
`
`
`
`
`
`mechanisms of cytotoxicity for each of the an-
`
`
`
`
`
`
`timetabolites. Major emphasis has also been placed
`
`
`
`
`on including pharmacokinetic and pharmacody-
`
`
`
`
`
`
`
`namic endpoints in clinical trials to help elucidate
`
`
`
`
`
`
`the optimal method of administration of single
`
`
`
`
`
`
`
`
`
`agents as well as the combination of two or more
`
`
`
`
`
`
`
`drugs. The various studies reviewed in this year’s
`
`
`
`
`
`
`chapter provide further insight into the mecha-
`nisms of action of the antimetabolites. As will be
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`described, emerging understanding of the biochem-
`
`
`
`
`
`
`ical and molecular determinants of drug sensitivity
`
`
`
`
`have provided new therapeutic strategies.
`
`2. Methotrexate
`
`
`
`
`
`
`Methotrexate (MTX) is a tight-binding inhibitor of
`
`
`
`
`
`
`the enzyme dihydrofolate reductase (DHFR), an es-
`
`
`
`
`
`sential enzyme in intracellular folate metabolism.
`
`
`
`
`
`
`
`DHFR is necessary for the conversion of dihydro-
`
`
`
`
`
`
`folate to tetrahydrofolate, and the reduced folates
`
`
`
`
`
`
`are. key "intermediates in one-carbon transfer reac-
`
`
`
`
`
`
`
`tions. An intact enyzme pathway is necessary to
`
`
`
`
`
`
`
`maintain de novo synthesis of purines and thymi-
`
`
`
`
`
`dine monophosphate (thymidylate). For this reason.
`
`
`
`
`
`
`DHFR represents a critical target enzyme in cancer
`chemotherapy.
`
`
`
`
`
`
`
`The precise mechanism(s) by which MTX ex-
`
`
`
`
`
`
`erts its cytotoxicity remains a subject of ongoing
`
`
`
`
`
`
`
`debate. The long-held belief has been that inhi-
`
`
`
`
`
`
`
`bition of DHFR by MTX leads to a depletion
`
`
`
`
`
`
`of intracellular levels of reduced folate cofactors,
`
`
`
`
`
`
`with subsequent impairment in de novo synthe-
`
`
`
`
`
`
`sis of purines and thymidylate. However, studies
`from several laboratories have demonstrated that
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`the level of reduced folates is depleted by only 50-
`
`
`
`
`
`
`70% (Annuals 10-17). Such a modest decrease in
`
`
`
`
`
`
`
`
`the reduced folate pool would seem to be insuf-
`
`
`
`
`
`
`ficient for the cytotoxic effects observed follow-
`
`
`
`
`
`
`
`
`ing treatment with MTX. It is now known that
`
`
`
`
`
`
`the polyglutamates of both MTX and dihydrofo-
`
`
`
`
`
`
`
`late, which accumulates in the presence of MTX-
`
`
`
`
`
`
`mediated DHFR blockade, are capable of directly
`
`
`
`
`
`inhibiting the activity of several folate-dependent
`
`
`
`
`
`
`enzymes in addition to DHFR, including thymidy-
`
`
`
`
`late synthase (TS), glycinamide ribonucleotide
`
`
`
`(GAR) trausformylase, and 5-aminoimidazo1e—4-
`
`
`
`carboxamide ribonucleotide (AICAR) transformy—
`
`
`
`
`
`
`lase. Thus, metabolic inhibition by MTX represents .
`
`
`
`
`
`
`a multifactorial process that involves partial de-
`
`
`
`
`
`
`
`pletion of key reduced folate substrates and direct
`
`
`
`
`inhibition of various foIate—dependent enzymes.
`
`
`
`
`[1]
`Fiskerstrand et al.
`investigated the ef-
`
`
`
`
`
`
`fects of MTX—mediated folate depletion on the
`
`
`
`
`activity of methionine synthase. This enzyme
`
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`
`Ch. I
`
`
`.I.L. Grem et al.
`
`
`
`
`
`
`
`Dose-dependent reductions in intracellular spermi-
`
`
`
`
`
`
`dine and sperrnine were noted, while putrescine
`was unaffected. Addition of either folinic acid
`
`
`
`
`
`
`
`
`
`or S-adenosyl-methionine prevented MTX-induced
`
`
`
`
`
`
`inhibition of polyamine synthesis. In contrast, in-
`
`
`
`
`cubation with hydrocoltisone or D-penicillamine,
`
`
`
`
`
`
`two other imrnunosuppressive agent used in the
`treatment of rheumatoid arthritis, had no effect on
`
`
`
`
`
`
`
`
`
`
`
`
`
`polyamine levels. These findings suggest that in-
`
`
`
`hibition of the S-adenosy1—methionine-dependent
`
`
`
`
`
`methyltransferase pathway by MTX interferes with
`
`
`
`
`
`
`spermidine and spermine synthesis in RA lympho-
`
`
`
`
`
`
`
`cytes. This effect may account for the immunosup-
`
`
`
`
`
`
`
`
`pressive action of MTX, but also may represent an
`
`
`
`
`
`
`alternative cytotoxic mechanism of MTX in ma-
`
`
`
`
`
`
`lignant cells that are especially dependent upon
`
`
`
`
`
`polyamine biosynthesis for growth and prolifera-
`tion.
`
`
`
`
`pre-
`systems,
`In several different model
`
`
`
`
`
`
`treatment of malignant cells with MTX results
`
`
`
`
`
`
`in the rapid intracellular accumulation of S-
`
`
`
`
`phosphoribosyl pyrophosphate. This effect has
`
`
`
`
`
`
`been exploited as a means to biochemically mod-
`
`
`
`
`
`
`ulate the antitumor activity of lluorouracil by
`
`
`
`
`
`
`enhancing its anabolism to the ribonucleotide level.
`
`
`
`
`
`However, studies using a chick fibroblast system
`
`
`
`
`
`
`suggest
`that PRPP may regulate the intracellu-
`
`
`
`
`
`
`lar synthesis of glucose transporters. With this
`
`
`
`
`
`
`
`in mind, Fung and colleagues [41 examined the
`
`
`
`
`potential relationship between MTX treatment,
`
`
`
`
`
`
`
`PRPP synthesis, and glucose transport as it re-
`
`
`
`
`
`
`lates to cytotoxicity in cultured Ehrlich ascites
`
`
`
`
`
`
`
`
`tumor cells. Treatment with up to 20 MM MTX
`resulted in a 2- to 3.5-fold increase in PRPP levels,
`
`
`
`
`
`
`
`
`
`
`
`
`
`accompanied by a significant suppression in the
`
`
`
`
`
`
`rate of glucose transport. Co—administration of 20
`
`
`
`
`
`lLM hypoxanthine with MTX completely protected
`
`
`
`
`
`
`
`against growth inhibition, and reversed the effect of
`
`
`
`
`
`
`
`
`MTX on PRPP production and the rate of glucose
`
`
`
`
`
`
`transport. Since glycolysis serves as a major energy
`
`
`
`
`
`
`supply for malignant cells, these findings suggest
`
`
`
`
`
`MTX-mediated inhibition of critical glucose trans-
`
`
`
`
`
`
`port mechanisms might starve cells of essential
`
`
`
`
`
`nutrients required to maintain cellular metabolism
`
`and growth.
`
`2 c
`
`
`
`
`
`
`
`atalyzes the transfer of a methyl group from
`
`
`5-methyltetrahydrofolate to homocysteine,
`thus
`
`
`
`
`
`forming methionine. Cobalamine is an important
`
`
`
`
`
`
`cofactor in this reaction pathway. Treatment of
`
`
`
`
`
`
`
`GaMg human glioma cells with _<_l ,u.M MTX re-
`
`
`
`
`
`
`sulted in a dose- and time-dependent reduction in
`
`
`
`
`
`both the total folate and 5-methyl-tetrahydrofolate
`
`
`
`
`
`
`
`
`
`pools in this cell
`line;
`the latter pool size was
`
`
`
`
`
`
`
`
`reduced by 50% at 3 h,
`and was barely de-
`
`
`
`
`
`
`tectable at 43 h. A significant dose-dependent
`
`
`
`
`
`
`
`reduction in the activity of methionine synthase co-
`
`
`
`
`
`
`incided with folate depletion. MTX also reduced
`
`
`
`
`the intracellular methyl—cobalamine content, pre-
`
`
`
`
`
`
`
`sumably the result of an inadequate supply of
`
`
`
`
`5-methyl-tetrahydrofolate cofactor required for re-
`
`
`
`
`
`methylation of homocysteine. Given that methion-
`
`
`
`
`
`
`ine synthase catalyzes a reaction-that involves re-
`
`
`
`
`
`
`duced folates, cobalamine, and sulfur amino acids,
`
`
`
`
`
`
`
`impaired function of this enzyme might have a
`
`
`
`
`
`
`significant impact on a number of critical ‘down-
`
`
`
`
`stream’ pathways, including transmethylation re-
`
`
`
`
`
`actions, polyamine synthesis, and protein biosyn-
`
`
`
`
`
`thesis. These findings suggest another potential
`
`
`
`cytotoxic mechanism for MTX.
`
`
`
`
`
`
`
`Schalinske and Steele [2] employed an in vivo
`
`
`
`
`
`
`
`
`rat model to investigate the effects of MTX on
`
`
`
`
`folate-dependent, one-carbon metabolism using a
`
`
`
`
`
`
`
`sensitive tracer kinetic method to quantify the car-
`
`
`
`
`
`
`bon flux through this pathway. Following a 7-day
`
`
`
`
`
`
`treatment with MTX, hepatic pools of tetrahydro-
`
`
`
`folate, 5—methyl-tetrahydrofolate, and 5-formy1-
`
`
`
`
`
`
`tetrahydrofolate were decreased by 63, 83 and
`
`
`
`
`
`58%, respectively. Compared to control animals,
`
`
`
`
`
`
`
`carbon flux through the one-carbon pool from his-
`
`
`
`
`
`
`tidine to methionine was significantly reduced by
`
`
`
`
`
`
`
`nearly 60% in MTX—treated rats. These kinetic ex-
`
`
`
`
`periments demonstrate MTX treatment markedly
`
`
`
`
`
`
`
`alters the actual carbon flow through the hepatic
`
`
`
`
`
`folate-dependent, one—carbon pool, and a major ef-
`fect is a reduction of carbon flow needed for the
`
`
`
`
`
`
`
`
`
`
`
`
`formation of both 5-methyl-tetrahydrofolate and
`methionine.
`
`
`
`
`
`
`
`
`Nesher et al. [3] investigated the in vitro effects
`
`
`
`
`
`
`
`of MTX on polyamine levels in lymphocytes ob-
`
`
`
`
`
`
`tained from patients with rheumatoid arthritis (RA).
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`

`
`Anrimetabalites Ch. I
`
`
`
`
`2.2. Falate transport
`
`
`
`
`
`
`
`
`Two major folate transport systems in human tis-
`sues have been well characterized at the molecular
`
`
`
`
`
`
`
`level. One is the classic reduced folate carrier
`
`
`
`
`
`
`
`
`
`
`
`
`(RFC) system,
`that has a relatively low affin-
`
`
`
`
`
`
`
`ity for reduced folates (affinity constants in the
`
`
`
`
`
`-micromolar range). However,
`the RFC system
`
`
`
`
`
`
`has a large capacity, and is primarily responsi-
`
`
`
`
`
`
`
`ble for MTX transport into cells at pharmaco-
`
`
`
`
`
`logical drug concentrations. The human reduced
`
`
`
`
`
`
`
`
`folate carrier gene has been mapped to the long
`
`
`
`
`
`
`
`arm of chromosome 21, and it encodes a pro-
`
`
`
`
`
`
`tein with a predicted molecular size of 59-68
`
`
`
`
`
`
`
`kDa (Annual 17). A second folate transport sys-
`
`
`
`
`tem involves a high-affinity membrane-bound, fo-
`
`
`
`
`
`
`late receptor binding protein (affinity constants for
`
`
`
`
`
`
`
`folic acid in the nanomolar range);
`this system
`
`
`
`
`
`
`
`has a much reduced capacity for transport of re-
`
`
`
`
`
`
`
`
`duced folates and MTX relative to the RFC sys-
`
`
`
`
`
`
`
`tem. The human folate receptor (FR) is a 38-40
`
`
`
`
`
`
`kDa glycoprotein bound to cellular membranes by
`
`
`a carboxyl-terrninal, glycosyl-phosphatidylinositol
`
`
`
`
`
`
`
`
`tail. This receptor is highly expressed on the sur-
`
`
`
`
`
`
`
`face of some epithelial tumors, such as ovarian
`
`
`
`
`
`
`cancer, making it a potentially useful target for
`
`
`
`
`
`antigen-directed anticancer therapies [5]. The hu-
`
`
`
`
`
`
`
`
`man FR appears to be the major transport mecha-
`
`
`
`
`
`
`
`nism for the uptake of non-classical antifols such
`
`
`
`as N10-propargyl-5,8—dideazafolic acid (CB37l7)
`
`
`
`and (6R)-5,10-dideaza-5,6,7,8-tetrahydrofolic acid
`
`(lometrexol, DDATI-IF).
`
`
`
`
`
`
`
`
`Because of the important role of the RFC in
`
`
`
`
`
`
`providing MTX transport,
`there is great interest
`
`
`
`
`
`
`
`
`
`in how the activity of the RFC gene is regulated.
`When cultured human leukemia CCRF-CEM cells
`
`
`
`
`
`
`
`
`
`
`
`
`were grown in folate-depleted media and then ex-
`
`
`
`
`
`
`
`posed to high concentrations of the reduced folate,
`
`
`
`
`
`leucovorin, down-regulation of the RFC protein
`
`
`
`
`
`
`
`
`on the cell surface was observed [6]. In contrast,
`
`
`
`
`
`addition of trimetrexate, a lipophilic DHFR in-
`
`
`
`
`
`hibitor, decreased intracellular reduced folate pools
`
`
`
`
`
`and blocked RFC down-regulation, suggesting that
`the relative size of the intracellular reduced folate
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`pool may be an important determinant of RFC ex-
`
`3
`
`
`
`
`
`
`
`
`pression. The regulation of RFC activity was also
`
`
`
`
`
`affected by other biochemical pathways dependent
`
`
`
`
`
`
`
`upon intracellular folates, such as de novo purine
`
`
`
`
`
`synthesis and DNA methylation reactions. Incu-
`bation of CCRF-CEM cells with either adenosine
`
`
`
`
`
`
`
`
`
`
`
`
`or S-adenosyl methionine also caused RFC down-
`
`
`
`
`
`regulation, but the underlying mechanism appeared
`
`
`
`
`
`
`
`to be independent of reduced folate pools. Thus,
`
`
`
`
`several diverse folate-dependent biochemical path-
`
`
`
`
`
`
`
`ways may contribute to the complex regulation of
`
`RFC expression.
`
`
`
`
`
`Relationships between MTX resistance and rel-
`
`
`
`
`
`
`
`ative RFC expression were explored further in stu'd-
`
`
`
`
`
`
`
`ies by Moscow et al.
`[7]. Transfection of RFC
`
`
`
`
`
`CDNA into a transport-deficient, MTX-resistant hu-
`man breast cancer line rendered them 250-fold
`
`
`
`
`
`
`more sensitive to MTX. The transfected cells were
`
`
`
`
`
`
`
`
`
`
`
`
`
`300-fold more resistant to trimetrexate, which en-
`
`
`
`
`
`
`
`ters cells through passive diffusion. The basis for
`trimetrexate resistance was atuibuted to enhanced
`
`
`
`
`
`
`
`
`
`
`
`
`uptake of reduced folates by the RFC system,
`
`
`
`
`
`
`which rescued cells from DHFR inhibition. Thus,
`
`
`
`
`
`
`
`increased expression of the RFC had disparate ef-
`
`
`
`
`
`
`
`fects on sensitivity to antifolates that utilize differ-
`
`
`
`
`ent mechanisms for cellular entry.
`
`
`
`
`
`Gorlick et al.
`[3] employed a flow cytome-
`
`
`
`
`
`
`
`. try method to assess MTX transport in_ human
`
`
`
`
`
`
`
`leukemic blast cells in which the competitive dis-
`
`
`
`
`
`placement of PT430, a fluorescent MTX analog.
`
`
`
`
`
`reflects reduced folate transport. Impaired MTX
`
`
`
`
`
`
`
`transport was observed in only 13% of untreated
`
`
`
`
`
`
`patients, compared with over 70% in patients
`
`
`
`
`
`who had relapsed following prior MTX-containing
`
`
`
`
`
`chemotherapy. Quantitation of RFC mRNA expres-
`
`
`
`
`
`
`sion indicated that impaired transport activity was
`
`
`
`
`
`associated with decreased RFC mR.NA expression,
`
`
`
`
`
`
`which (supports reduced transport capacity as an
`
`
`
`
`
`important mechanism of clinical MTX resistance.
`
`
`
`
`
`The molecular changes responsible for MTX-
`
`
`
`
`resistance was examined in a transport—deficient
`
`
`
`
`
`
`
`human T—cell lymphoblastic cell line [9]. No differ-
`
`
`
`
`
`
`
`ence in RFC mRNA levels by reverse transcription-
`
`
`
`
`polymerase chain reaction (RT-PCR) methodol-
`
`
`
`
`
`ogy was noted compared with parental wild-type,
`
`
`MTX-sensitive cells. However, nucleotide se-
`
`
`
`--.--a----a-av-.-———.——-..__..—.........-—...-.—V\.....
`
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`
`

`
`Ch. I
`
`
`
`.I.L. Grem er al.
`
`
`
`
`
`
`
`
`
`The genomic structure of the RFC gene in Chi-
`
`
`
`
`
`
`
`nese hamster ovary cells was reported by Murray
`
`
`
`
`
`
`
`
`et al. [13]. The RFC localized to hamster chromo-
`
`
`
`
`
`
`
`
`some 1, and contained seven exons and six introns
`
`
`
`
`
`
`
`extending over a range of 15.3 kb. Two alterna-
`
`
`
`
`
`
`tively spliced mRNA products were detected in
`
`
`
`
`
`
`these cells, however, the functional significance of
`
`
`
`
`
`these splicing variants requires further study.
`At least three different isoforms of the human
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`FR exist and are subclassified as FR-0.‘, FR—l3, and
`
`
`
`
`
`
`FR-y (Annual 17). These isoforms have different
`
`
`
`
`
`
`folate binding affinities, and variable expression in
`
`
`
`
`
`
`
`specific tissues. FR-or is highly expressed in hu-
`
`
`
`
`
`
`
`man epithelial tissues and in some cancers, while
`
`
`
`
`
`
`
`
`
`FR-,8 is found in the human placenta and other non-
`
`
`
`
`
`epithelial tissues. Human FR-y lacks a glycosyl-
`
`
`
`
`
`phosphatidylinositol membrane anchor and is most
`
`
`
`
`
`
`likely a secretory protein. Alterations in tissue ex-
`
`
`
`
`
`
`
`
`
`pression of the FR can be induced by changes in
`
`
`
`
`
`
`exogenous folate concentration, or by alterations in
`
`
`
`
`
`
`normal physiology, such as during pregnancy. The
`
`
`
`
`
`
`
`complete sequence of the FR-or promoter in human
`
`
`
`
`
`
`KB nasophalyngeal cancer cells was recently pub-
`
`
`
`
`
`
`
`lished by Elwood and colleagues [14]. Variations in
`
`
`
`
`
`
`the 5’-terminus of human FR-or mRNA transcripts
`
`
`
`
`
`
`
`
`were found to result from two separate and unique
`
`
`
`
`
`
`
`
`upstream promoter sites, one in exon 1, and the
`
`
`
`
`
`
`
`other in exon 4. Additional heterogeneity of the
`
`
`
`
`
`
`
`human FR-o: mRNA was also caused by differen-
`
`
`
`
`
`
`tial mRNA spicirlg events involving the 5’-exons.
`
`
`
`
`
`
`The biological consequences of this genomic com-
`
`
`
`
`
`
`
`plexity are not known, but may potentially play
`
`
`
`
`
`a role in post—transcriptional regulation. Support-
`
`
`
`
`
`
`
`
`ive evidence for this theory is provided by Roberts
`
`
`
`
`
`
`
`
`
`et al. [15] who showed that differences in the 5’-
`
`
`
`
`
`
`
`untranslated region of the human FR-or mRNA can
`
`
`
`
`
`
`greatly affect the efficiency of protein synthesis.
`
`
`
`
`
`
`Furthermore, both cis- and trans-acting control el-
`ements have been identified in the 5’-untranslated
`
`
`
`
`
`
`
`
`
`
`
`
`
`region of human FR—a: mRNA that specifically reg-
`
`
`
`
`
`
`ulate human FR translational efficiency [16]. Ad-
`ditional studies of the translational control of the
`
`
`
`
`
`
`
`
`
`
`
`human FR expression are warranted.
`
`
`
`
`
`
`The protein structures of human FR-or and
`
`
`
`
`
`
`
`FR-,8 were examined by Shen et al.
`[17] using
`
`4 q
`
`
`
`
`
`
`
`
`uencing of the resistant cells identified two dif-
`
`
`
`
`
`
`
`ferent premature stop codons in the RFC allele,
`
`
`
`
`
`
`thus providing an explanation for the decreased
`
`
`
`
`
`
`RFC protein expression. These findings support the
`
`
`
`
`
`
`
`importance of the RFC in influencing MTX sen-
`
`
`
`
`
`
`
`sitivity, but also highlight the danger of relying
`
`
`
`
`
`
`
`
`solely on mRNA levels to screen for reduced RFC
`
`transport capacity.
`
`
`
`
`
`
`
`
`Wong et a1. [10] found that transfection of hu-
`
`
`
`
`
`man RFC cDNA into a transport-deficient human
`
`
`
`
`
`K562 erythroleukemia cell line enhanced cellular
`
`
`
`
`
`
`uptake of MTX. However, protein affinity label-
`
`
`
`
`
`
`ing experiments revealed that only a small portion
`
`
`
`
`
`
`
`
`of the RFC protein on the cell membrane was
`
`
`
`
`
`
`functionally active. Other factors, as yet uncharac-
`
`
`
`
`
`
`terized, were postulated to influence the regulation
`
`
`
`
`
`
`
`of RFC activity. Transfection of RFC cDNA into
`
`
`
`
`
`
`transport-deficient L1 2 10 murine leukemia cells in-
`
`
`
`
`
`
`
`
`
`creased rate of MTX influx, but resulted in only a
`
`
`
`
`
`
`modest net change in intracellular MTX concentra-
`
`
`
`
`
`
`
`tion [11]. Further studies revealed that the increase
`in RFC-mediated influx was counterbalanced by an
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`increase in MTX effiux out of the cell by a sep-
`
`
`
`
`
`
`arate transport system. MTX cytotoxicity in these
`
`
`
`
`
`
`
`A cells was therefore influenced more strongly by the
`
`
`
`
`
`
`net change in MTX drug accumulation. Bidirec-
`
`
`
`
`
`
`
`tional changes in MTX transport thus appear to
`
`
`
`
`
`
`be important in determining the overall therapeutic
`
`
`
`
`
`
`consequences of changes in RFC gene expression,
`
`
`
`
`
`
`and these findings illustrate that increased expres-
`
`
`
`
`
`
`
`
`
`sion of RFC protein may not always translate into a
`
`
`
`
`corresponding increase in MTX accumulation.
`
`
`
`
`
`
`
`Folate transport activity can be difficult to mea-
`
`
`
`
`
`
`sure directly in freshly obtained clinical specimens.
`
`
`
`
`
`
`
`A new, simplified method was developed by Jolivet
`
`
`
`
`
`
`
`
`et al. [12] that may allow more widespread testing
`
`
`
`
`
`of RFC-mediated transport activity. Using confo-
`
`
`
`
`
`cal microscopic visualization. the accumulation of
`
`
`
`
`
`fiuorescently tagged MTX analogs was measured
`within the cell. Intracellular fluorescence correlated
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`with RFC activity both in _tumor cell lines and in
`leukemic blasts obtained from children with acute
`
`
`
`
`
`
`
`
`
`
`
`lymphoblastic leukemia. This method holds great
`
`
`
`
`
`
`
`
`
`promise because of the relative ease in which it can
`
`
`
`
`be applied to clinical specimens.
`
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`
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`
`

`
`Antimetabolites Ch. I
`
`
`
`
`
`
`
`
`chimeric constructs arising from both receptor sub-
`
`
`
`
`
`
`types. These isoforms share about 70% amino
`
`
`
`
`
`
`
`acid sequence homology, but differ in their stereo-
`
`
`
`
`
`
`specificity for reduced folate binding. The human
`
`
`
`
`
`
`FR-or has a relative higher binding affinity for
`
`
`
`
`the physiological (6S)-reduced folate stereoisomer
`
`
`
`
`
`
`
`than FR-33. The major structural changes in human
`
`
`
`
`
`
`FR-oz responsible for this difference were localized
`to a leucine for alanine substitution at amino acid
`
`
`
`
`
`
`
`
`
`
`
`
`
`position 49, and to additional differences down-
`
`
`
`
`
`
`
`
`stream of residue 92. Further studies are in progress
`
`
`
`
`
`
`
`
`to better define the precise changes in amino acid
`
`
`
`
`
`
`sequence responsible for the alterations in isoform
`
`binding affinity.
`
`
`
`
`
`
`Other differences in human FR activity may
`
`
`
`
`
`
`
`be caused by structural changes in the recep-
`
`
`
`tor’s glycosyl-phosphatidylinositol membrane an-
`
`
`
`
`
`chor.
`the tail of which is a post-translational
`
`
`
`
`
`polypeptide modification found on a number of
`
`
`
`
`functionally diverse membrane proteins. Glycosyl-
`
`
`
`
`phosphatidylinositol-anchored proteins can be en-
`
`
`
`
`
`
`
`zymatically released from the cell surface by the
`
`
`
`activity of a phosphatidylinositol-specific phospho-
`
`
`
`
`
`
`
`lipase. Wang et al. [18] recently characterized two
`murine L12l0 leukemic cell sublines that both ex-
`
`
`
`
`
`
`
`
`
`
`
`
`pressed membrane-bound FR-or, but differed in
`
`
`
`
`
`
`
`their folate binding affinity by 17-fold. The FR
`
`
`
`
`
`
`
`amino acid structure and the mRNA coding se-
`
`
`
`
`
`
`
`
`quence in the two cell lines were identical, and
`
`
`
`
`
`
`
`the only structural difference was localized to the
`
`
`
`
`FR glycosyl-phosphatidylinositol tail. Indirect ev-
`
`
`
`
`
`
`idence suggested that different fatty acyl substitu-
`
`
`
`
`
`
`
`
`tions on the inositol ring were responsible for the
`
`
`
`
`
`
`variation in receptor binding affinity. Thus, func-
`
`
`
`
`
`
`
`
`tional changes in the FR activity may arise from
`
`
`
`
`differences in the post-translational modifications.
`
`
`
`
`
`
`independent of the underlying amino acid structure.
`
`
`
`
`
`Membrane components may also influence hu-
`
`
`
`
`
`
`man FR activity. Depletion of membrane choles-
`
`
`
`
`
`
`
`terol has been reported to inhibit human FR-
`
`
`
`
`
`
`
`mediated transport (Annual 15). Stevens et al [19]
`
`
`
`
`
`recently found that membrane sphingolipid content
`
`
`
`
`
`
`affected human FR-mediated uptake in colon ade-
`nocarcinoma CaCo-2 cells. Administration of the
`
`
`
`
`
`
`
`
`
`
`mycotoxin fumonisin B1 blocked the synthesis of
`
`5
`
`
`
`
`
`membrane sphingolipids, effectively shutting down
`
`
`
`
`
`
`human FR-mediated transport. The total amount of
`human FR on the cell surface decreased in fumon-
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`isin B1-treated cells, while other receptor-mediated
`
`
`
`
`
`transport functions were unaffected, suggesting a
`
`
`
`
`
`
`relatively specific effect. The authors caution that
`
`
`
`
`
`
`the ingestion of fungally contaminated food prod-
`
`
`
`
`
`
`
`ucts by pregnant women might potentially lead to
`
`
`
`
`
`
`
`an acquired folate deficiency and be responsible for
`folate-related birth defects.
`
`
`
`
`
`
`
`
`
`
`The clinical importance of the human FR in the
`
`
`
`
`
`cellular transport of newer, non-classical, antifolate
`
`
`
`
`
`
`drugs was examined by Pinard et al.
`[20]. Two
`
`MTX-resistant human breast cancer cell lines in-
`
`
`
`
`
`
`
`
`
`
`
`
`creased their human FR mRNA expression when
`
`
`
`
`
`
`adapted for growth in low folate-containing me-
`
`
`
`
`
`
`dia. One cell line, MTXR-ZR-75-1, predominantly
`
`
`
`
`
`
`expressed human FR-oz, while MDA-231 cells ex-
`
`
`
`
`
`
`pressed human FR-,6. Both lines lacked functional
`
`
`
`
`RFC-mediated transport. The increased expression
`
`
`
`
`
`
`
`
`of human FR in both lines corresponded to a
`
`
`
`
`
`
`
`180-400-fold increased sensitivity to CB37l7 and
`
`
`
`
`
`
`
`lometrexol, consistent with the high affinity of the
`
`
`
`
`
`
`
`FR for these newer antifolate agents. In contrast,
`
`
`
`
`
`
`drug sensitivity to MTX, edatrexate, and raltitrexed
`
`
`
`
`
`(Tomudexm), was only slightly increased, sug-
`
`
`
`
`
`
`
`gesting that the FR was not a major transporter
`
`
`
`
`
`
`for these agents under the experimental conditions.
`
`
`
`
`
`
`
`Thus, when the RFC system is non-functional, the
`
`
`
`
`
`
`
`
`amount of human FR activity may be an impor-
`
`
`
`
`
`
`tant determinant of cellular sensitivity to newer
`
`
`
`
`
`
`antifolates with high affinity for this receptor.
`
`
`
`
`
`The relationship between FR expression and
`
`
`
`
`
`
`lometrexol toxicity was examined in greater de-
`
`
`
`
`
`
`
`
`tail by Sen et al. [21]. Lometrexol, which blocks
`
`
`
`
`
`
`
`de novo purine synthesis by inhibiting GAR trans-
`
`
`
`
`
`
`
`forrnylase,
`is an excellent substrate for the FR
`
`
`
`
`
`
`system. Transfection of human FR-oz cDNA into
`
`
`
`
`
`
`NIH/3T3 cells increased drug uptake, making them
`10-fold more sensitive to lometrexol. This relation-
`
`
`
`
`
`
`
`
`
`
`
`
`ship was examined further in four non-transfected
`
`
`
`
`
`
`
`
`human ovarian cancer cell lines with high basal ex-
`
`
`
`
`
`
`
`
`pression of human FR-at. All four cell lines were
`
`
`
`
`
`
`sensitive to lometrexol, but the relative sensitivity
`
`
`
`
`
`
`
`
`did not correlate with the cell surface density of
`
`Lilly Ex. 2076
`Sandoz V. Lilly IPR20l6-00318
`
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`Lilly Ex. 2076
`Sandoz v. Lilly IPR2016-00318
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`Ch. I
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`J.L. Grem etal.
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`mechanism termed potocytosis has been proposed
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`for FR-mediated uptake into cells, which pos-
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`tulates that the folate ligand—-receptor complexes’
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`cluster within specific invaginations of the mem-
`brane. These caveolae are distinct from*clatl1rin-
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`coated pits involved in classic receptor-mediated
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`endocytosis, and the invaginations can ‘pinch’ off
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`and thus internalize the ligand——receptor complex.
`Acidification of the enclosed vesicles then causes
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`dissociation of the receptor complex, followed by
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`folate uptake across the membrane into the cy-
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`toplasm. This mechanism accounts for the rapid
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`kinetics of the FR recycling on the cell surface.
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`Most of the supporting evidence for potocytosis
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`comes from morphological experiments showing
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`clustering of the FR in caveolae, although some
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`reports have questioned whether this represents a
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`fixation procedure artifact (Annuals 15 and 16). Us-
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`ing fluorescence spectroscopy and pH—dependent,
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`fluorescently tagged folic acid probes, Lee et a1.
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`[24] measured the pH of internalized membrane
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`vesicles containing FR complexes taken up from
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`the cell surface. The intraluminal pH was found
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`to be low, ranging from 4.7 to 5.8, consistent with
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`the acidification step predicted by the potocytosis
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`mechanism. Smart and colleagues [25] used a cell
`fractionation method to confirm that FRs cluster
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`within caveolae on the surface of monkey kidney
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`MA104 cells, consistent with the proposed poro-
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`cytosis mechanism. Wu et al. [26] examined FR
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`clustering on the cell surface using monovalent flu-
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`orescently tagged probes to prevent cross—linking
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`[26]. Two cell lines with only modest expression
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`of the FR, epithelial JAR cell