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Commentary on “Thymidylate Synthase Pharmacogenetics in Colorectal Cancer”
Commentary on “Thymidylate Synthase Pharmacogenetics in Colorectal Cancer” M. Sitki Copur
Figure 1: Fluoropyrimidine Metabolic Pathway
Saint Francis Cancer Center Grand Island, Nebraska
Catabolism (85%)
Edward Chu Department of Medicine and Pharmacology, Yale Cancer Center, Yale University School of Medicine and VA CT Cancer Center West Haven, CT
FUrd
FU
NMP Kinase
UK
UP
DPD FUH2
RNA
OPRTase NDP Kinase FUDP
FUMP
FUTP
TP
The discovery of the link between genetic polymorphisms in drug-metabolizing enzymes, transporters, receptors, and other drug targets and the interindividual differences observed in the clinical efficacy and toxicity of many medications has created a new field, termed pharmacogenetics. This area of research has generated significant interest and excitement in the medical profession and the pharmaceutical industry. Pharmacogenetics has been traditionally used to define a relatively narrow spectrum of inherited genetic disorders in drug metabolism and disposition. However, it is clear that the ability of a given drug to exert a certain pharmacologic effect requires the interaction of the drug with a specific cell surface receptor, enzyme, and/or target protein. The ability of this initial drug-target interaction to then activate certain critical intracellular pathways involved in signal transduction, cell cycle control, or apoptosis might ultimately determine whether a given drug is able to exert its biologic effect. During the past few years, intense research efforts have focused on the area of pharmacogenomics. In general, this field of science investigates the potential importance of genetic variation as a critical determinant of drug efficacy and/or toxicity.1,2 The fluoropyrimidine, 5-fluorouracil (5FU), was first synthesized in the late 1950s, and is a widely used anticancer agent with activity against a broad range of human malignancies. Despite having been in clinical practice for such a long period of time, the biochemical and molecular mechanisms by which 5-FU exerts its cytotoxic and host toxic effects are complex and involve several critical pathways, as outlined in Figure 1.3-7 There are several lines of evidence to support the view that the folate-dependent enzyme thymidylate synthase (TS) is one of the important targets for 5-FU. This enzyme catalyzes the reaction, which provides the sole intracellular de novo source of thymidylate, an essential metabolite for DNA biosynthesis. While a genetic polymorphism has been identified for TS, it would not be surprising if other polymorphisms in
Ribonucleotide Reductase
FdUrd FUPA NMP Kinase
TK FdUMP
NDP Kinase FdUDP
FdUTP
dUTP Hydrolase
FBAL TS
dTMP
dUMP 5,10-CH2THF
DNA
DHF
Abbreviations: 5,10-CH2THF = 5,10-methylene tetrahydrofolate; DHF = dihydrofolate; DPD = dihydropyrimidine; dTMP = 2'-deoxythymidine-5'-monophosphate; dUMP = deoxyuridine monophosphate; dUTP Hydrolase = deoxyuridine triphosphate; FBAL = fluoro-β-alanine; FdUDP = fluorodeoxyuridine diphosphate; FdUMP = 5-fluoro-2'-deoxyuridine monophosphate; FdUrd = 5-fluoro-2'-deoxyuridine; FdUTP = fluorodeoxyuridine triphosphate; FU = fluorouracil; FUDP = 5-fluorouridine-5'-diphosphate; FUH2 = dihydro-5'-fluorouracil; FUMP = 5-fluorouridine monophosphate; FUPA = α-fluoro-β-ureido-propanoic acid; FUrd = 5-fluorouridine; FUTP = 5-fluoro-2'-deoxyuridine triphosphate; NDP Kinase = nucleoside diphosphate kinase; NMP Kinase = nucleoside monophosphate kinase; OPRTase = orotate phosphoribosyltransferase; TK = thymidine kinase; TP = thymidine phosphorylase; TS = thymidylate synthase; UK = uridine kinase; UP = uridine phosphorylase
genes involved in the various pathways outlined in Figure 1 were identified, including those relating to the activation of 5-FU to the cytotoxic nucleotide metabolites, those relating to the degradation of 5-FU, such as dihydropyrimidine dehydrogenase (DPD) or other nucleotidases, and those involved in DNA repair such as dUTP nucleotidohydrolase (dUTPase) and the mismatch repair class of enzymes. As an additional layer of complexity, the exact mechanism by which TS enzyme inhibition ultimately results in cell death remains to be more clearly defined, and it might ultimately depend upon the expression of critical cellular signaling proteins involved in cell cycle checkpoint control and apoptosis. Finally, the control mechanisms regulating the expression of TS are extremely complex, and include control at the level of transcription, posttranscription, translation, and posttranslation.8 There is growing evidence that multiple levels of control might be at play, depending on the cellular context and milieu and the various factors surrounding exposure to a given
genotoxic and/or cytotoxic stress such as dose, schedule, and timing of drug administration. As a result, it remains to be established whether a polymorphism in the TS gene can actually result in alterations in expression of TS protein and/or TS enzyme activity. Clearly, further molecular-based investigations are required to confirm such a mechanistic link. It is now well established that the response to a given drug is often a polygenic trait. Pharmacogenomic studies are mainly focused on elucidating the inherited nature of genetic polymorphisms causing interindividual differences in drug effects and metabolism. However, such polymorphisms might not have direct clinical relevance, depending on the molecular basis of the polymorphism, the expression of other key drugmetabolizing enzymes, the presence of concurrent medications or illnesses, and/or other polygenic clinical features that impact upon response. Further studies are required to define the true biological and clinical relevance of such pharmacogenomic traits.
Clinical Colorectal Cancer November 2001
• 179
In this issue of Clinical Colorectal Cancer, Marsh and McLeod9 present a thorough and well-written review of TS pharmacogenetics. This review is especially timely, as there is now growing evidence documenting the importance of TS as a chemotherapeutic target and a fairly strong correlation between the level of TS expression and fluoropyrimidine chemosensitivity. However, there are several important issues that are raised by this review and by the entire field of pharmacogenomics. First, while a correlation can be made between the presence of a given genetic polymorphism, does such a correlation exist because of a true mechanistic link or does it exist simply as a result of statistical significance? Unfortunately, this must always be considered when dealing with so-called prognostic molecular biomarkers. Second, does the genetic polymorphism exist only in malignant tissue or can it also be observed in normal tissue? If so, would it then be possible to identify a surrogate normal tissue to test more easily for the presence of the genetic polymorphism in healthy individuals as well as in cancer patients? Finally, does a given genetic polymorphism exist as a single genetic event or is it merely representative of an entire constellation of genetic abnormalities resulting from the genomic instability found in nearly all human malignancies?
With the recent advances in molecular sequencing technology, the identification of genetic polymorphisms might give us only an initial glimpse of their potential role as a marker for drug action. This first step must be followed by more precise preclinical molecular-based studies to confirm and establish that these genomic polymorphisms indeed have a true biologic effect. Once the biologic function is established, it will then be important to extend these studies to the clinical setting to show that these polymorphisms can be translated into true phenotypic consequences in patients. The rapid advances in biotechnology with DNA microarray systems, tissue microarrays, highthroughput screening systems, and advanced bioinformatics, along with the data emerging from the Human Genome Project, should permit the development and application of therapeutic agents for specific genetically identifiable subgroups of cancer patients.10 The availability of rapid, relatively inexpensive, valid, and reliable technology for genetic testing and molecular profiling of a patient’s tumor will be essential as we begin the dramatic shift from traditional empiric chemotherapy to patient-specific tailored therapy. 01. Kalow W. Pharmacogenetics in biological perspective. Pharmacol Rev 1997; 49:369-379.
180 • Clinical Colorectal Cancer November 2001
02. Kalow W. Pharmacogenetics, pharmacogenomics, and pharmacobiology. Clin Pharmacol Ther 2001; 70:1-4. 03. Daher GC, Harris BE, Diasio RB. Metabolism of pyrimidine analogues and their nucleosides. Pharmacol Ther 1990; 48:189-222. 04. Grem J. 5-Fluoropyrimidines. In: Chabner B, Long D, eds. Cancer Chemotherapy and Biotherapy. 2nd ed. Philadelphia: Lippincott-Raven, 1996:149-212. 05. Milano G, Etinne M. Fluorinated pyrimidines. In: Grochow L, Ames M, eds. Clinician's Guide to Chemotherapy Pharmacokinetics and Pharmacodynamics. Baltimore: Williams & Wilkins, 1998:289300. 06. Allegra C, Grem J. Antimetabolites. In: De Vita V, Hellman S, Rosenberg S, eds. Cancer Principles and Practice of Oncology. 5th ed. Philidelphia: Lippincott-Raven, 1997:432-451. 07. Moran RG, Spears CP, Heidelberger C. Biochemical determinants of tumor sensitivity to 5-fluorouracil: ultrasensitive methods for the determination of 5fluoro-2'-deoxyuridylate, 2'-deoxyuridylate, and thymidylate synthetase. Proc Natl Acad Sci U S A 1979; 76:1456-1460. 08. Chu E, Ju J, Schmitz J. Molecular regulation of expression of thymidylate synthase. In: Jackman A, ed. Anticancer Drug Development Guide: Antifolate Drugs in Cancer Therapy. Totowa: Humana Press, 1999:397-408. 09. Marsh S, McLeod L. Thymidylate synthase pharmacogenetics in colorectal cancer. Clin Colorectal Cancer 2001; 1:175-178. 10. Evans WE, Relling MV. Pharmacogenomics: translating functional genomics into rational therapeutics. Science 1999; 286:487-491.
Figure 1: Fluoropyrimidine Metabolic Pathway
Saint Francis Cancer Center Grand Island, Nebraska
Catabolism (85%)
Edward Chu Department of Medicine and Pharmacology, Yale Cancer Center, Yale University School of Medicine and VA CT Cancer Center West Haven, CT
FUrd
FU
NMP Kinase
UK
UP
DPD FUH2
RNA
OPRTase NDP Kinase FUDP
FUMP
FUTP
TP
The discovery of the link between genetic polymorphisms in drug-metabolizing enzymes, transporters, receptors, and other drug targets and the interindividual differences observed in the clinical efficacy and toxicity of many medications has created a new field, termed pharmacogenetics. This area of research has generated significant interest and excitement in the medical profession and the pharmaceutical industry. Pharmacogenetics has been traditionally used to define a relatively narrow spectrum of inherited genetic disorders in drug metabolism and disposition. However, it is clear that the ability of a given drug to exert a certain pharmacologic effect requires the interaction of the drug with a specific cell surface receptor, enzyme, and/or target protein. The ability of this initial drug-target interaction to then activate certain critical intracellular pathways involved in signal transduction, cell cycle control, or apoptosis might ultimately determine whether a given drug is able to exert its biologic effect. During the past few years, intense research efforts have focused on the area of pharmacogenomics. In general, this field of science investigates the potential importance of genetic variation as a critical determinant of drug efficacy and/or toxicity.1,2 The fluoropyrimidine, 5-fluorouracil (5FU), was first synthesized in the late 1950s, and is a widely used anticancer agent with activity against a broad range of human malignancies. Despite having been in clinical practice for such a long period of time, the biochemical and molecular mechanisms by which 5-FU exerts its cytotoxic and host toxic effects are complex and involve several critical pathways, as outlined in Figure 1.3-7 There are several lines of evidence to support the view that the folate-dependent enzyme thymidylate synthase (TS) is one of the important targets for 5-FU. This enzyme catalyzes the reaction, which provides the sole intracellular de novo source of thymidylate, an essential metabolite for DNA biosynthesis. While a genetic polymorphism has been identified for TS, it would not be surprising if other polymorphisms in
Ribonucleotide Reductase
FdUrd FUPA NMP Kinase
TK FdUMP
NDP Kinase FdUDP
FdUTP
dUTP Hydrolase
FBAL TS
dTMP
dUMP 5,10-CH2THF
DNA
DHF
Abbreviations: 5,10-CH2THF = 5,10-methylene tetrahydrofolate; DHF = dihydrofolate; DPD = dihydropyrimidine; dTMP = 2'-deoxythymidine-5'-monophosphate; dUMP = deoxyuridine monophosphate; dUTP Hydrolase = deoxyuridine triphosphate; FBAL = fluoro-β-alanine; FdUDP = fluorodeoxyuridine diphosphate; FdUMP = 5-fluoro-2'-deoxyuridine monophosphate; FdUrd = 5-fluoro-2'-deoxyuridine; FdUTP = fluorodeoxyuridine triphosphate; FU = fluorouracil; FUDP = 5-fluorouridine-5'-diphosphate; FUH2 = dihydro-5'-fluorouracil; FUMP = 5-fluorouridine monophosphate; FUPA = α-fluoro-β-ureido-propanoic acid; FUrd = 5-fluorouridine; FUTP = 5-fluoro-2'-deoxyuridine triphosphate; NDP Kinase = nucleoside diphosphate kinase; NMP Kinase = nucleoside monophosphate kinase; OPRTase = orotate phosphoribosyltransferase; TK = thymidine kinase; TP = thymidine phosphorylase; TS = thymidylate synthase; UK = uridine kinase; UP = uridine phosphorylase
genes involved in the various pathways outlined in Figure 1 were identified, including those relating to the activation of 5-FU to the cytotoxic nucleotide metabolites, those relating to the degradation of 5-FU, such as dihydropyrimidine dehydrogenase (DPD) or other nucleotidases, and those involved in DNA repair such as dUTP nucleotidohydrolase (dUTPase) and the mismatch repair class of enzymes. As an additional layer of complexity, the exact mechanism by which TS enzyme inhibition ultimately results in cell death remains to be more clearly defined, and it might ultimately depend upon the expression of critical cellular signaling proteins involved in cell cycle checkpoint control and apoptosis. Finally, the control mechanisms regulating the expression of TS are extremely complex, and include control at the level of transcription, posttranscription, translation, and posttranslation.8 There is growing evidence that multiple levels of control might be at play, depending on the cellular context and milieu and the various factors surrounding exposure to a given
genotoxic and/or cytotoxic stress such as dose, schedule, and timing of drug administration. As a result, it remains to be established whether a polymorphism in the TS gene can actually result in alterations in expression of TS protein and/or TS enzyme activity. Clearly, further molecular-based investigations are required to confirm such a mechanistic link. It is now well established that the response to a given drug is often a polygenic trait. Pharmacogenomic studies are mainly focused on elucidating the inherited nature of genetic polymorphisms causing interindividual differences in drug effects and metabolism. However, such polymorphisms might not have direct clinical relevance, depending on the molecular basis of the polymorphism, the expression of other key drugmetabolizing enzymes, the presence of concurrent medications or illnesses, and/or other polygenic clinical features that impact upon response. Further studies are required to define the true biological and clinical relevance of such pharmacogenomic traits.
Clinical Colorectal Cancer November 2001
• 179
In this issue of Clinical Colorectal Cancer, Marsh and McLeod9 present a thorough and well-written review of TS pharmacogenetics. This review is especially timely, as there is now growing evidence documenting the importance of TS as a chemotherapeutic target and a fairly strong correlation between the level of TS expression and fluoropyrimidine chemosensitivity. However, there are several important issues that are raised by this review and by the entire field of pharmacogenomics. First, while a correlation can be made between the presence of a given genetic polymorphism, does such a correlation exist because of a true mechanistic link or does it exist simply as a result of statistical significance? Unfortunately, this must always be considered when dealing with so-called prognostic molecular biomarkers. Second, does the genetic polymorphism exist only in malignant tissue or can it also be observed in normal tissue? If so, would it then be possible to identify a surrogate normal tissue to test more easily for the presence of the genetic polymorphism in healthy individuals as well as in cancer patients? Finally, does a given genetic polymorphism exist as a single genetic event or is it merely representative of an entire constellation of genetic abnormalities resulting from the genomic instability found in nearly all human malignancies?
With the recent advances in molecular sequencing technology, the identification of genetic polymorphisms might give us only an initial glimpse of their potential role as a marker for drug action. This first step must be followed by more precise preclinical molecular-based studies to confirm and establish that these genomic polymorphisms indeed have a true biologic effect. Once the biologic function is established, it will then be important to extend these studies to the clinical setting to show that these polymorphisms can be translated into true phenotypic consequences in patients. The rapid advances in biotechnology with DNA microarray systems, tissue microarrays, highthroughput screening systems, and advanced bioinformatics, along with the data emerging from the Human Genome Project, should permit the development and application of therapeutic agents for specific genetically identifiable subgroups of cancer patients.10 The availability of rapid, relatively inexpensive, valid, and reliable technology for genetic testing and molecular profiling of a patient’s tumor will be essential as we begin the dramatic shift from traditional empiric chemotherapy to patient-specific tailored therapy. 01. Kalow W. Pharmacogenetics in biological perspective. Pharmacol Rev 1997; 49:369-379.
180 • Clinical Colorectal Cancer November 2001
02. Kalow W. Pharmacogenetics, pharmacogenomics, and pharmacobiology. Clin Pharmacol Ther 2001; 70:1-4. 03. Daher GC, Harris BE, Diasio RB. Metabolism of pyrimidine analogues and their nucleosides. Pharmacol Ther 1990; 48:189-222. 04. Grem J. 5-Fluoropyrimidines. In: Chabner B, Long D, eds. Cancer Chemotherapy and Biotherapy. 2nd ed. Philadelphia: Lippincott-Raven, 1996:149-212. 05. Milano G, Etinne M. Fluorinated pyrimidines. In: Grochow L, Ames M, eds. Clinician's Guide to Chemotherapy Pharmacokinetics and Pharmacodynamics. Baltimore: Williams & Wilkins, 1998:289300. 06. Allegra C, Grem J. Antimetabolites. In: De Vita V, Hellman S, Rosenberg S, eds. Cancer Principles and Practice of Oncology. 5th ed. Philidelphia: Lippincott-Raven, 1997:432-451. 07. Moran RG, Spears CP, Heidelberger C. Biochemical determinants of tumor sensitivity to 5-fluorouracil: ultrasensitive methods for the determination of 5fluoro-2'-deoxyuridylate, 2'-deoxyuridylate, and thymidylate synthetase. Proc Natl Acad Sci U S A 1979; 76:1456-1460. 08. Chu E, Ju J, Schmitz J. Molecular regulation of expression of thymidylate synthase. In: Jackman A, ed. Anticancer Drug Development Guide: Antifolate Drugs in Cancer Therapy. Totowa: Humana Press, 1999:397-408. 09. Marsh S, McLeod L. Thymidylate synthase pharmacogenetics in colorectal cancer. Clin Colorectal Cancer 2001; 1:175-178. 10. Evans WE, Relling MV. Pharmacogenomics: translating functional genomics into rational therapeutics. Science 1999; 286:487-491.