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Myeloprotection with drug-resistance genes
Review
Myeloprotection with drug-resistance genes
Myeloprotection with drug-resistance genes Debabrata Banerjee and Joseph R Bertino
One of the many applications of gene transfer for cancer gene therapy is the transfer of drug-resistance genes into bone-marrow stem cells for myeloprotection. Protection of the hosts' bone marrow should allow for dose escalation that may be useful for eradicating minimal residual disease in a post-transplant situation. A number of drug resistance genes, whose products include mutant forms of enzymes that confer resistance to chemotherapeutic drugs, are discussed. Advances in hematopoietic stem cell isolation and ex vivo manipulation has kept pace with improvements in retroviral vector technology to make hematopoietic stem cell transduction a distinct reality. Clinical trials, which have established that the approach is safe, are now being designed to address more therapeutically relevant issues.
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Gene therapy can be defined as the introduction and expression of an exogenous gene with the aim of correcting inherited or acquired disease.1–3 Replacement of a defective gene with a functional one is a simple and straightforward concept. The practicalities, such as choice of vectors for targeting particular cell types, long-term expression, safety issues, undesired immune responses, and cost of treatment, are continually being investigated, and new and improved guidelines are being drawn up. In cancer gene therapy,4–7 the intentions are to restore a missing tumour-suppressor gene,8–11 inactivate a rogue gene,12,13 increase the antitumour immune response by oncolytic viruses,14–16 and activate the immune stimulatory mechanisms.17–21 Strategies can include the transfer of a drug-resistance gene to haemopoietic stem cells to protect the recipient from myelotoxicity of chemotherapy, especially after transplantation.
Transfer of drug-resistance genes into haemopoietic progenitors for myeloprotection A potentially important use of gene-transfer technology is to transform bone-marrow progenitor cells (Figure 1) into a drug-resistant state, which might allow conventional or larger doses of chemotherapeutic agents to be administered safely after transplantation without a risk of severe myelosuppression.22 Patients in whom conventional treatments fail – eg those with large-cell lymphoma – are given high-dose regimens of drug combinations and are rescued with reinfusion of stored autologous marrow or peripheral-blood stem cells (PBSCs). This approach has increased the number of complete remissions and cure rates in lymphoma and germ-cell tumours, but still some patients 154
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Figure 1. A scanning electron micrograph of bone marrow stem cells.
relapse.23 Furthermore, the approach may not be beneficial for patients with breast cancer,24 although arguments can be made in favour of myeloprotection for high-dose chemotherapy after transplantation of gene-modified stem cells. Sensitivity of the patient’s haemopoietic system immediately after high-dose chemotherapy and transplantation of haemopoietic stem cells has discouraged DB and JRB were members of the Molecular Pharmacology and Experimental Therapeutics Program, Sloan Kettering Institute for Cancer Research, New York, until December 2001, and are now at the Cancer Institute of New Jersey, Departments of Medicine and Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey. Correspondence: Dr D Banerjee, Cancer Institute of New Jersey, Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ 08903, USA. Tel: +1 732 235 6458. Fax: +1 732 235 8181. Email [email protected]
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Review
Myeloprotection with drug-resistance genes
the use of post-transplant chemotherapy. Successful transfer of drug-resistance genes to haemopoietic progenitors might allow post-transplant treatment with appropriate chemotherapeutic agents, but is yet to be tested in the clinic as a therapeutic approach. Clinical trials designed to assess the safety of transduction should be followed by trials that measure the therapeutic benefit of this approach, which can be monitored at the molecular level through continued expression of the drug-resistance gene. Treatment of a genetic disease requires that the transferred gene be incorporated into the genome of a stem cell so as to remain with the individual for life. By contrast, gene therapy as applicable to malignant disease will probably require only transient expression of the desired protein, perhaps into committed progenitor cells; thus, this goal may be more achievable.
Collect PBSC
Isolate stem cells
Stimulate with cytokines
For purposes of myeloprotection, the CD34-positive PBSCs seem to be the ideal target cells. These cells are isolated from peripheral blood by means of commercial columns that rely on either positive selection with CD34 antibody or negative selection by elimination of cells that do not express the CD34 antigen. The US Food and Drug Administration has already approved one of these devices for isolating CD34positive PBSCs for clinical use and may consider approving another system. At present, the vector of choice for gene transfer into PBSCs is oncoretroviruses, although lentiviruses have also been considered because they may be more efficient in transduction of PBSCs. Since retroviral transduction occurs in dividing cells, the isolated PBSCs need to be stimulated to enter the cell cycle, but not to differentiate. After isolation, the PBSCs are treated in vitro with several growth factors, including stem-cell factor, interleukins 3 and 6, thrombopoietin, Flt-3, and granulocyte-macrophage colony-stimulating factor. Stemcell factor (c-kit), thrombopoietin, and Flt-3 ligand may be sufficient for inducing progenitor cells to divide without differentiation. Figure 2 illustrates the process from PBSCs harvest, retroviral infection, integration, transplantation, and treatment with chemotherapeutic drugs.
Retroviral vectors Many of the retroviral vectors that have been used for transducing PBSCs have been derived from the murine Moloney leukaemia virus. The vector backbone has been modifed to increase expression of transgenes in haemopoietic cells such as the MPSV promoter enhancer element.25 A major problem in sustained transgene expression has been gene silencing, probably due to promoter methylation. Several investigators have attempted to overcome this problem to maintain transgene expression in transduced cells. One approach was to include insulator elements in the vector backbone, and this modification seems to decrease methylation.26 Other considerations in optimisation of gene expression include message stability and translational efficiency of the message. Modifications in the long terminal repeat region of the retroviral vector, such as removal of splice acceptor sites and inclusion of enhancer elements, result in improved gene expression.27 Elements of THE LANCET Oncology Vol 3 March 2002
Infect with retrovirus containing drug-resistance genes
Drug resistance gene
Chemotherapy
Return transduced cells
Illustration by Terry Helms, Medical Graphics, Memorial Sloan-Kettering Cancer Center
Target cells
Figure 2. Process of PBSC harvest, retroviral infection, integration, transplantation, and treatment with chemotherapeutic drugs.
viral internal ribosomal entry sites (IRES) are commonly used to express two or more genes from one transcriptional unit, but this approach generally results in low expression of the downstream gene in relation to the first. The impaired expression of the second gene can be circumvented by the use of fusion genes, provided that they are small genes, that both are functional when expressed, and that they do not have restricted subcellular localisations.
Drug-resistance genes The best characterised drug-resistance genes are mutant forms of the genes encoding dihydrofolate reductase (DHFR) and the DNA repair enzyme O6-methylguanine transferase (MGMT), and the multidrug resistance gene (MDR1). Mutant forms of DHFR confer resistance to methotrexate, a widely used antifolate. Several different mutants of both the human and the rodent gene have been reported. Some, with substitution of arginine or phenylalanine for leucine at residue 22 or of serine or
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Myeloprotection with drug-resistance genes
Table 1. Drug-resistance genes and chemotherapeutic agents that may be useful in myelosuppression Drug-resistance gene
Chemotherapeutic agent
DHFR, mutant forms
Antifolates (methotrexate, trimetrexate)
Thymidylate synthase, mutant forms
Fluorouracil, folate inhibitors (raltitrexed, thymitaq)
MDR1
Anthracyclines, vinca alkaloids, paclitaxel, etoposide
GST
Mechlorethamine
Cytidine deaminase/ 5’ nucleotidase
Nucleoside analogues (cytarabine, gemcitabine)
ALDH
Cyclophosphamide
Alkylguanine DNA alkyl Nitrosoureas (carmustine, temozolomide) transferases (such as MGMT)
tryptophan for phenylalanine at residue 31, as well as double mutants such as Phe22/Ser31, have been used as dominant selectable markers in cultured cells as well as for genetransfer studies in vivo.28–31 In a study by May and colleagues,35 protection of recipient mice by reconstitution with mutant DHFR-transduced bone-marrow cells also protected the structure of the microvilli in the gut epithelium of recipients when challenged with methotrexate. The explanation for this effect is not yet known. Drug-resistant mutants of the human and the bacterial thymidylate synthase gene have been described.32–35 The cDNAs when transfected into cells confer resistance to inhibitors of thymidylate synthase, such as fluorouracil and thymitaq. The G52S mutant human thymidylate synthase gene (wild-type glycine at 52 mutated to serine) confers up to 97-fold resistance to floxuridine and up to 12-fold resistance to thymitaq. This mutant should prove useful in myeloprotection strategies because fluorouracil is commonly used in the treatment of breast and colon cancers. We have begun to examine the possibility of expressing more than one drug-resistance gene to protect against a combination of chemotherapeutic drugs. These genes may be expressed under the control of a single promoter to circumvent the issues of differing transcriptional efficiencies of different promoters or may be expressed from the same promoter but as separate translational units by the use of IRES elements. A fusion gene encoding a double mutant of human DHFR and cytidine deaminase when transfected into mammalian cells protects them from both methotrexate and cytarabine.36 Others have constructed vectors containing MDR1 and alkylguanine-DNA alkyltransferases to confer resistance against several chemotherapeutic agents for myeloprotection strategies.41 We have constructed a fusion gene from the double mutant of DHFR (the F/S DHFR) and a mutant form of human thymidylate synthase (G52S) to confer dual resistance to methotrexate and fluorouracil.38 This particular construct, along with the MDR1 gene or the ALDH gene, may be useful in myeloprotection strategies in breast cancer, for which combination chemotherapy with cyclophosphamide, fluorouracil, and methotrexate or doxorubicin is used. A drug-resistance gene already in clinical trial is MDR1,39–41 which encodes a 170 kDa membrane protein (pglycoprotein) that effluxes various drugs, mostly natural 156
products, thereby lowering the intracellular concentration of the drug and conferring resistance against a wide variety of structurally unrelated drugs. Some normal tissues and cell types also express p-glycoprotein, for example, the gastrointestinal mucosa and the epithelial cells of the choroid plexus. Previously, haemopoietic stem cells were thought to be resistant to drugs affected by this process only because they were not in the cycling phases of the cell cycle, but recent evidence suggests that p-glycoprotein expression in these cells may also contribute to the resistance phenotype. To confer resistance to antifolates, a mutant DHFR cDNA has to be introduced; by contrast, expression of only the wild-type MDR gene is sufficient to cause multidrug resistance. Gene-transfer studies to confer the MDR phenotype to the haemopoietic stem cells have shown that the transduced cells can exclude the panel of MDR drugs and thus become resistant. Clinical trials of retroviral vectors containing the MDR1 gene have been modestly successful at best. Although low gene expression was detectable for as long as a year after transplantation, the gene was truncated possibly owing to abnormal splicing. Improvements in gene-transfer methods (eg use of fibronectin, and the use of thrombopoietin in the cytokine cocktail) have allowed more efficient gene transfer. Although myelodysplasia has been observed in mice reconstituted with bone-marrow cells transduced with MDR1,42 this feature has not been seen in patients enrolled in clinical trials.39 An important enzyme that contributes to increased cellular resistance to nitrosoureas is MGMT. This enzyme prevents the generation of DNA interstrand cross-links induced by the chloroethylnitrosoureas by reacting with the cross-link precursors O6-chloroethylguanine or O6,N1ethanoguanine monoadducts in DNA and transferring the alkyl lesion to an internal cysteine residue.43 Moritz and colleagues44 reported that transduction of murine bonemarrow cells with a retroviral construct containing the MGMT gene confers resistance to carmustine. Infusion of MGMT-transduced bone-marrow progenitor cells to mice protected them from the toxic effects of weekly intraperitoneal injections of 40 mg/kg, strongly supporting the contention that gene transfer of MGMT may be useful for protection of the haemopoietic system from high-dose nitrosourea therapy. A recent development has been the generation of mutant forms of MGMT that are not sensitive to O6-benzylguanine, an inhibitor of this enzyme. Thus, transduced bone-marrow progenitor cells are protected from the toxicity of O6-benzylguanine and carmustine, whereas the tumour cells that contain the wild-type enzyme are sensitive.5,44 Table 1 summarises the various drugresistance genes that may be of use in myeloprotection and the chemotherapeutic agents against which resistance is conferred.28–31
Other gene products conferring drug resistance Genes involved in the antioxidant defence pathway, such as BCL2 and those encoding glutathione-associated enzymes, also confer drug resistance. The BCL2 (B-cell lymphoma/leukaemia 2) proto-oncogene was originally discovered because it is involved in t(14;18) translocations
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Myeloprotection with drug-resistance genes
Review
Conclusions Search strategy and selection criteria The literature search for this review was done on line with PubMed and the following search terms: ‘gene transfer’, ‘drug resistance’, and ‘myeloprotection’ from 1980 onwards for publications in English. Those provided as guides to further reading do not represent the entire literature on the subject and we apologise to the investigators whose contributions were not listed owing to space constraints. common to many non-Hodgkin lymphomas. It is an antiapoptotic protein. The t(14;18) translocations place the BCL2 gene at 18q21 in juxtaposition to the immunoglobulin heavy-chain locus at 14q32, resulting in transcriptional deregulation of BCL2. Induction of apoptosis by such external stimuli as radiation, hyperthermia, growth-factor withdrawal, glucocorticoids, and many classes of chemotherapeutic agents, is inhibited by BCL2 in vitro. Gene-transfer studies involving transfection of BCL2 cDNA into cells has unequivocally shown that cells transfected with, and overexpressing, this gene become resistant to various chemotherapeutic agents. Cells overexpressing BCL2 generate peroxides, but their cellular constituents including lipid membranes are not damaged. In mice, retrovirusmediated gene transfer of the BCL2 gene into bone-marrow cells protects the transplanted marrow cells from chemotherapy-induced myelosuppression without compromising the antitumour activity of the administered chemotherapeutic agent. Reconstitution of mice with these transduced marrow cells resulted in complete myeloprotection, compared with controls, when challenged with a myelosuppressive drug. Long-term adverse effects of BCL2 overexpression have not been studied,49 however, and such studies are needed. Glutathione-associated enzymes are important in cellular detoxification of xenobiotics. The glutathione-Stransferase (GST) enzyme family is particularly important with respect to anticancer-drug resistance. Four major isoforms of cytosolic GST have been described in mammalian cells. They share homology and are therefore thought to belong to the GST supergene family.50,51 The GST Π and the GST-Yc forms have been implicated in cellular resistance to nitrogen mustards and nitrosoureas, respectively. Transfection of the GST isoform cDNAs into breast-cancer cells (MCF7) did not produce convincing resistance to anticancer drugs. However, MCF7 cells have one of the lowest intrinsic GST-specific activities and thus have an inherently low capacity for synthesis of reduced glutathione (GSH). Therefore, in these cells, the GSH pathway may not be an important detoxification mechanism. Transfection of rat GST-Yc gene into rat mammary tumour cells and transfection of cDNAs of other GST enzymes into yeast and Chinese hamster ovary cells resulted in significantly higher resistance to anticancer drugs such as nitrogen mustards, cisplatin, and carboplatin. Gene transfer of the rat Yc gene into NIH 3T3 mouse fibroblasts and into the haemopoietic system in vitro resulted in an increase of five to ten fold in resistance to chlorambucil and mechlorethamine.51 THE LANCET Oncology Vol 3 March 2002
Cancer gene therapy has come a long way, from the euphoria of early unrealistic promises to the present-day realities. Obstacles such as poor efficiency of gene transfer, lack of tumour specificity, loss of sustained expression, and undesired toxic effects need to be addressed. Fresh hope comes from the recent success in retrovirus-mediated gene transfer and expression of the γC cytokine receptor subunit and subsequent correction of the severe combined immunodeficiency phenotype in two infants52 and correction of haemophilia due to deficiency of factor IX by transfer of the factor IX gene with adenoassociated virus.53 These successes are encouraging, but much remains to be done. As with any form of new treatment, cancer gene therapy, including myeloprotection strategies, has to undergo rigorous clinical trials of efficacy and long-term safety. Transfer and expression of drug-resistance genes in stem cells of cancer patients should theoretically permit safer dosing and dose-intensive chemotherapy. Future clinical trials should include poor-risk patients with largecell lymphoma, testicular cancer, and choriocarcinoma, who are likely to benefit from such an approach. Patients with small-cell lung cancer, or breast, ovarian, or bladder cancer may also benefit. Phase II trials should examine the possibility that there is an improved therapeutic index compared with historical controls, and phase III trials should compare post-transplant treatment (tolerance and efficacy) in patients transplanted with and without drugresistance genes. Acknowledgments
DB and JRB are supported by grants from the National Cancer Institute: CA-59350, CA-61586, and CA-86438. References
1 Verma IM, Somia N. Gene therapy: promises, problems and prospects. Nature 1997; 389: 239–42. 2 Somia N, Verma IM. Gene therapy: trials and tribulations. Nat Rev Genet 2000; 1: 91–99. 3 Romano G, Micheli P, Pacilio C, et al. Latest developments in gene transfer technology: achievements, perspectives, and controversies over therapeutic applications. Stem Cells 2000; 18: 19–39. 4 Ribas A, Butterfield LH, Economou JS. Genetic immunotherapy for cancer. Oncologist 2000; 5: 87–98. 5 Lattime EC, Gerson SL, (Eds).Gene therapy of cancer: translational approaches from preclinical studies to clinical implementation. San Diego: Academic Press, 2000. 6 Nagy A, Habib S. Cancer gene therapy: past achievements and future challenges. In: Advances in experimental medicine and biology, vol 465. New York: Kluwer Academic/Plenum, 2000. 7 Vile RG, Russell SJ, Lemoine NR. Cancer gene therapy: hard lessons and new courses. Gene Therapy 2000; 7: 2–8. 8 Roth J, Mukhopadhyay T, Zhang WW, et al. Gene replacement strategies for lung cancer. Semin Radiat Oncol 1996: 6: 105–09. 9 Claudio PP, Howard CM, Pacilio C, et al. Mutations in the retinoblastoma-related gene RB2/p130 in lung tumors and suppression of tumor growth in vivo by retrovirus mediated gene transfer. Cancer Res 2000; 60: 372–82. 10 Frost SJ, Simpson DJ, Clayton RN, et al. Transfection of an inducible p16/CDKN2A construct mediates reversible growth inhibition and G1 arrest in the AtT20 pituitary tumor cell line. Mol Endocrinol 1999; 13: 1801–10. 11 Yang CT, You L, Chang JW, et al. Adenovirus-mediated p14(ARF) gene transfer in human mesothelioma cells. J Natl Cancer Inst 2000; 92: 636–41. 12 Jansen B, Wacheck V, Heere-Ress E, et al. Chemosensitisation of malignant melanoma by BCL2 antisense therapy. Lancet 2000; 356: 1728–33.
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13 Galderisi U, Cascino A, Giordano A. Antisense oligonucleotides as therapeutic agents. J Cell Physiol 1999; 181: 251–57. 14 Mineta T, Rabkin SD, Yazaki T, et al. Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas. Nat Med 1995; 1: 938–43. 15 Bischoff,JR. Kirn DH, Williams A, et al. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science 1996; 274: 373–76. 16 Fueyo J, Gomez-Manzano C, Alemany R, et al. A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo. Oncogene 2000; 19: 2–12. 17 Timmerman JM, Levy R. Dendritic cell vaccines for cancer immunotherapy. Annu Rev Med 1999; 50: 507–29. 18 Boczkowski D, Nair SK, Snyder D, et al. Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. J Exp Med 1996; 184: 465–72. 19 Kumar V, Sercarz E. Genetic vaccination: the advantages of going naked. Nat Med 1996; 2: 857–59. 20 Seder RA, Gurunathan S. DNA vaccines–designer vaccines for the 21st century. N Engl J Med 1999; 341: 277–78. 21 Clay TM, Custer MC, Sachs J, et al. Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity. J Immunol 1999; 163: 507–13. 22 Bertino JR. “Turning the tables”– making normal marrow resistant to chemotherapy. J Natl Cancer Inst 1990; 82: 1234–35. 23 Gianni AM, Bregni M, Sena S, et al. Recombinant human granulocyte macrophage stimulating factor reduces hematologic toxicity and widens clinical applicability of high dose cyclophosphamide treatment in breast cancer and non-Hodgkin’s lymphoma. J Clin Oncol 1990; 8: 768–78. 24 Hudis C, Fornier M, Riccio L, et al. 5-year results of dose-intensive sequential adjuvant chemotherapy for women with high-risk nodepositive breast cancer: a phase II study. J Clin Oncol 1999; 17: 1118–24. 25 Riviere I, Brose K, Mulligan RC. Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells. Proc Natl Acad Sci USA 1995; 92: 6733–37. 26 Rivella S, Callegari JA, May C, et al. The cHS4 insulator increases the probability of retroviral expression at random chromosomal integration sites. J Virol 2000; 74: 4679–86. 27 Hildinger M, Abel KL, Ostertag W, Baum C. Design of 5' untranslated sequences in retroviral vectors developed for medicinal use. J Virol 1999; 73: 4083–91. 28 Corey CA, DeSilva AD, Holland CA, Williams DA. Serial transplantation of methotrexate resistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells. Blood 1990; 76: 337–43. 29 Zhao SC, Li MX, Banerjee D, et al. Long term protection of recipient mice from lethal doses of methotrexate by marrow infected with a double copy retrovirus containing a mutant dihydrofolate reductase . Cancer Gene Ther 1994; 1: 27–33. 30 Allay JA, Persons DA, Galipeau J, et al. In vivo selection of retrovirally transduced hematopoietic stem cells. Nat Med 1998; 4: 1136–43. 31 May C, Gunther R, McIvor RS. Protection of mice from lethal doses of methotrexate by transplantation with transgenic bone marrow expressing drug resistant dihydrofolate reductase activity. Blood 1995; 86: 2439–48. 32 Barbour KW, Berger SH, Berger FG. A single amino acid substitution defines a naturally occurring genetic variant of human thymidylate synthase. Mol Pharmacol 1990; 37: 515–18. 33 Tong Y, Liu-Chen X, Ercikan-Abali EA, et al. Isolation and characterization of Thymitaq(AG337) and 5-fluoro-2deoxyuridylate-resistant mutants of human thymidylate synthase from ethylmethanesulfonate-exposed human sarcoma HT-1080 cells. J Biol Chem 1998; 273: 11611–18. 34 Landis DM, Loeb LA. Random sequence mutagenesis and resistance
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to 5-fluorouridine in human thymidylate synthases. J Biol Chem 1998; 273: 25809–17. Fantz C, Shaw D, Jennings W, et al. Drug-resistant variants of Escherichia coli thymidylate synthase: effects of substitutions at Pro254. Mol Pharmacol 2000; 57: 359–66. Sauerbrey A, McPherson JP, Zhao SC, et al. Expression of a novel double mutant dihydrofolate reductase-cytidine deaminase fusion gene confers resistance to both methotrexate and cytosine arabinoside. Hum Gene Ther 1999; 10: 2495–504. Jelinek J, Rafferty JA, Cmejla R, et al. A novel dual function retrovirus expressing multidrug resistance 1 and O6-alkylguanineDNA-alkyltransferase for engineering resistance of haemopoietic progenitor cells to multiple chemotherapeutic agents. Gene Ther 1999; 6: 1489–93. Capiaux GM, Budak-Alpdogan T, Takebe N, Banerjee D, Bertino JR. A novel fusion protein that confers resistance to both methotrexate and 5-fluorouracil. Proc Am Assoc Cancer Res 2001; 42: 29 (abstr 155). Abonour R, Williams DA, Einhorn L, et al. Efficient retrovirusmediated transfer of multidrug resistance-1 gene into autologous human long-term repopulating hematopoietic stem cells. Nat Med 2000; 6: 652–58. Moscow JA, Huang H, Carter C, et al. Engraftment of MDR1 and NeoR gene-transduced hematopoietic cells after breast cancer chemotherapy. Blood 1999; 94: 52–61. Hesdorffer C, Ayello J, Ward M, et al. Phase I trial of retroviralmediated transfer of the human MDR1 gene as marrow chemoprotection in patients undergoing high-dose chemotherapy and autologous stem-cell transplantation. J Clin Oncol 1998; 16: 165–72. Bunting KD, Galipeau J, Topham D, et al. Transduction of murine bone marrow cells with an mdr-1 vector enables ex vivo stem cell expansion, but these expanded grafts cause a myeloproliferative syndrome in transplanted mice. Blood 1998; 92: 2269–79. Pegg AE, Weist L, Foote RS, et al. Purification and properties of O6methylguanine DNA transmethylase from rat liver. J Biol Chem 1983; 258: 2327–33. Moritz T, Mackay W, Glassner BJ, et al. Retrovirus-mediated expression of a DNA repair protein in bone marrow protects hematopoietic cells from nitrosourea-induced toxicity in vitro and in vivo. Cancer Res 1995; 55: 2608–14. Banerjee D, Zhao SC, Li M-X, et al. Gene therapy utilizing drug resistant genes: a review. Stem Cells 1994; 12: 378–84. McIvor RS. Drug resistant dihydrofolate reductases: generation, expression and therapeutic application. Bone Marrow Transplant 1999; 18: S50–54. Blakley RL, Sorrentino BP. In vitro mutations in dihydrofolate reductase that confer resistance to methotrexate: potential for clinical application. Hum Mutat 1998; 11: 259–65. Licht T, Herrmann F, Gottesman MM, Pastan I. In vivo drug selectable genes: a new concept in gene therapy. Stem Cells 1997; 15: 104–11. Kondo S, Yin D, Morimura T, et al. Transfection with a bcl-2 expression vector protects transplanted bone marrow from chemotherapy induced myelosuppression. Cancer Res 1994; 54: 2928–33. Daniel V. Glutathione-S-transferase: gene structure and regulation of expression. Crit Rev Biochem Mol Biol 1993; 28: 173–207. Letourneau S, Greenbaum M, Cournoyer D. Retrovirus mediated gene transfer of rat glutathione S-transferase Yc confers in vitro resistance to alkylating agents in human leukemic cells and in clonogenic mouse hematopoietic progenitor cells. Hum Gene Ther 1996; 7: 831–40. Cavazzana-Calvo M, Hacein-Bey S, de SaintBasile G, et al. Gene therapy of human severe combined immunodeficiency (SCID)-XI disease. Science 2000; 288: 669–72. Kay MA, Manno CS, Ragni MV, et al. Evidence for gene transfer and expression of factor IX in hemophilia B patients treated with an AAV vector. Nat Genet 2000; 24: 257–61.
THE LANCET Oncology Vol 3 March 2002
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Myeloprotection with drug-resistance genes
Myeloprotection with drug-resistance genes Debabrata Banerjee and Joseph R Bertino
One of the many applications of gene transfer for cancer gene therapy is the transfer of drug-resistance genes into bone-marrow stem cells for myeloprotection. Protection of the hosts' bone marrow should allow for dose escalation that may be useful for eradicating minimal residual disease in a post-transplant situation. A number of drug resistance genes, whose products include mutant forms of enzymes that confer resistance to chemotherapeutic drugs, are discussed. Advances in hematopoietic stem cell isolation and ex vivo manipulation has kept pace with improvements in retroviral vector technology to make hematopoietic stem cell transduction a distinct reality. Clinical trials, which have established that the approach is safe, are now being designed to address more therapeutically relevant issues.
Rights were not granted to include this image in electronic media. Please refer to the printed journal.
Gene therapy can be defined as the introduction and expression of an exogenous gene with the aim of correcting inherited or acquired disease.1–3 Replacement of a defective gene with a functional one is a simple and straightforward concept. The practicalities, such as choice of vectors for targeting particular cell types, long-term expression, safety issues, undesired immune responses, and cost of treatment, are continually being investigated, and new and improved guidelines are being drawn up. In cancer gene therapy,4–7 the intentions are to restore a missing tumour-suppressor gene,8–11 inactivate a rogue gene,12,13 increase the antitumour immune response by oncolytic viruses,14–16 and activate the immune stimulatory mechanisms.17–21 Strategies can include the transfer of a drug-resistance gene to haemopoietic stem cells to protect the recipient from myelotoxicity of chemotherapy, especially after transplantation.
Transfer of drug-resistance genes into haemopoietic progenitors for myeloprotection A potentially important use of gene-transfer technology is to transform bone-marrow progenitor cells (Figure 1) into a drug-resistant state, which might allow conventional or larger doses of chemotherapeutic agents to be administered safely after transplantation without a risk of severe myelosuppression.22 Patients in whom conventional treatments fail – eg those with large-cell lymphoma – are given high-dose regimens of drug combinations and are rescued with reinfusion of stored autologous marrow or peripheral-blood stem cells (PBSCs). This approach has increased the number of complete remissions and cure rates in lymphoma and germ-cell tumours, but still some patients 154
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Lancet Oncol 2002; 3: 154–58
Figure 1. A scanning electron micrograph of bone marrow stem cells.
relapse.23 Furthermore, the approach may not be beneficial for patients with breast cancer,24 although arguments can be made in favour of myeloprotection for high-dose chemotherapy after transplantation of gene-modified stem cells. Sensitivity of the patient’s haemopoietic system immediately after high-dose chemotherapy and transplantation of haemopoietic stem cells has discouraged DB and JRB were members of the Molecular Pharmacology and Experimental Therapeutics Program, Sloan Kettering Institute for Cancer Research, New York, until December 2001, and are now at the Cancer Institute of New Jersey, Departments of Medicine and Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey. Correspondence: Dr D Banerjee, Cancer Institute of New Jersey, Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ 08903, USA. Tel: +1 732 235 6458. Fax: +1 732 235 8181. Email [email protected]
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Review
Myeloprotection with drug-resistance genes
the use of post-transplant chemotherapy. Successful transfer of drug-resistance genes to haemopoietic progenitors might allow post-transplant treatment with appropriate chemotherapeutic agents, but is yet to be tested in the clinic as a therapeutic approach. Clinical trials designed to assess the safety of transduction should be followed by trials that measure the therapeutic benefit of this approach, which can be monitored at the molecular level through continued expression of the drug-resistance gene. Treatment of a genetic disease requires that the transferred gene be incorporated into the genome of a stem cell so as to remain with the individual for life. By contrast, gene therapy as applicable to malignant disease will probably require only transient expression of the desired protein, perhaps into committed progenitor cells; thus, this goal may be more achievable.
Collect PBSC
Isolate stem cells
Stimulate with cytokines
For purposes of myeloprotection, the CD34-positive PBSCs seem to be the ideal target cells. These cells are isolated from peripheral blood by means of commercial columns that rely on either positive selection with CD34 antibody or negative selection by elimination of cells that do not express the CD34 antigen. The US Food and Drug Administration has already approved one of these devices for isolating CD34positive PBSCs for clinical use and may consider approving another system. At present, the vector of choice for gene transfer into PBSCs is oncoretroviruses, although lentiviruses have also been considered because they may be more efficient in transduction of PBSCs. Since retroviral transduction occurs in dividing cells, the isolated PBSCs need to be stimulated to enter the cell cycle, but not to differentiate. After isolation, the PBSCs are treated in vitro with several growth factors, including stem-cell factor, interleukins 3 and 6, thrombopoietin, Flt-3, and granulocyte-macrophage colony-stimulating factor. Stemcell factor (c-kit), thrombopoietin, and Flt-3 ligand may be sufficient for inducing progenitor cells to divide without differentiation. Figure 2 illustrates the process from PBSCs harvest, retroviral infection, integration, transplantation, and treatment with chemotherapeutic drugs.
Retroviral vectors Many of the retroviral vectors that have been used for transducing PBSCs have been derived from the murine Moloney leukaemia virus. The vector backbone has been modifed to increase expression of transgenes in haemopoietic cells such as the MPSV promoter enhancer element.25 A major problem in sustained transgene expression has been gene silencing, probably due to promoter methylation. Several investigators have attempted to overcome this problem to maintain transgene expression in transduced cells. One approach was to include insulator elements in the vector backbone, and this modification seems to decrease methylation.26 Other considerations in optimisation of gene expression include message stability and translational efficiency of the message. Modifications in the long terminal repeat region of the retroviral vector, such as removal of splice acceptor sites and inclusion of enhancer elements, result in improved gene expression.27 Elements of THE LANCET Oncology Vol 3 March 2002
Infect with retrovirus containing drug-resistance genes
Drug resistance gene
Chemotherapy
Return transduced cells
Illustration by Terry Helms, Medical Graphics, Memorial Sloan-Kettering Cancer Center
Target cells
Figure 2. Process of PBSC harvest, retroviral infection, integration, transplantation, and treatment with chemotherapeutic drugs.
viral internal ribosomal entry sites (IRES) are commonly used to express two or more genes from one transcriptional unit, but this approach generally results in low expression of the downstream gene in relation to the first. The impaired expression of the second gene can be circumvented by the use of fusion genes, provided that they are small genes, that both are functional when expressed, and that they do not have restricted subcellular localisations.
Drug-resistance genes The best characterised drug-resistance genes are mutant forms of the genes encoding dihydrofolate reductase (DHFR) and the DNA repair enzyme O6-methylguanine transferase (MGMT), and the multidrug resistance gene (MDR1). Mutant forms of DHFR confer resistance to methotrexate, a widely used antifolate. Several different mutants of both the human and the rodent gene have been reported. Some, with substitution of arginine or phenylalanine for leucine at residue 22 or of serine or
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Table 1. Drug-resistance genes and chemotherapeutic agents that may be useful in myelosuppression Drug-resistance gene
Chemotherapeutic agent
DHFR, mutant forms
Antifolates (methotrexate, trimetrexate)
Thymidylate synthase, mutant forms
Fluorouracil, folate inhibitors (raltitrexed, thymitaq)
MDR1
Anthracyclines, vinca alkaloids, paclitaxel, etoposide
GST
Mechlorethamine
Cytidine deaminase/ 5’ nucleotidase
Nucleoside analogues (cytarabine, gemcitabine)
ALDH
Cyclophosphamide
Alkylguanine DNA alkyl Nitrosoureas (carmustine, temozolomide) transferases (such as MGMT)
tryptophan for phenylalanine at residue 31, as well as double mutants such as Phe22/Ser31, have been used as dominant selectable markers in cultured cells as well as for genetransfer studies in vivo.28–31 In a study by May and colleagues,35 protection of recipient mice by reconstitution with mutant DHFR-transduced bone-marrow cells also protected the structure of the microvilli in the gut epithelium of recipients when challenged with methotrexate. The explanation for this effect is not yet known. Drug-resistant mutants of the human and the bacterial thymidylate synthase gene have been described.32–35 The cDNAs when transfected into cells confer resistance to inhibitors of thymidylate synthase, such as fluorouracil and thymitaq. The G52S mutant human thymidylate synthase gene (wild-type glycine at 52 mutated to serine) confers up to 97-fold resistance to floxuridine and up to 12-fold resistance to thymitaq. This mutant should prove useful in myeloprotection strategies because fluorouracil is commonly used in the treatment of breast and colon cancers. We have begun to examine the possibility of expressing more than one drug-resistance gene to protect against a combination of chemotherapeutic drugs. These genes may be expressed under the control of a single promoter to circumvent the issues of differing transcriptional efficiencies of different promoters or may be expressed from the same promoter but as separate translational units by the use of IRES elements. A fusion gene encoding a double mutant of human DHFR and cytidine deaminase when transfected into mammalian cells protects them from both methotrexate and cytarabine.36 Others have constructed vectors containing MDR1 and alkylguanine-DNA alkyltransferases to confer resistance against several chemotherapeutic agents for myeloprotection strategies.41 We have constructed a fusion gene from the double mutant of DHFR (the F/S DHFR) and a mutant form of human thymidylate synthase (G52S) to confer dual resistance to methotrexate and fluorouracil.38 This particular construct, along with the MDR1 gene or the ALDH gene, may be useful in myeloprotection strategies in breast cancer, for which combination chemotherapy with cyclophosphamide, fluorouracil, and methotrexate or doxorubicin is used. A drug-resistance gene already in clinical trial is MDR1,39–41 which encodes a 170 kDa membrane protein (pglycoprotein) that effluxes various drugs, mostly natural 156
products, thereby lowering the intracellular concentration of the drug and conferring resistance against a wide variety of structurally unrelated drugs. Some normal tissues and cell types also express p-glycoprotein, for example, the gastrointestinal mucosa and the epithelial cells of the choroid plexus. Previously, haemopoietic stem cells were thought to be resistant to drugs affected by this process only because they were not in the cycling phases of the cell cycle, but recent evidence suggests that p-glycoprotein expression in these cells may also contribute to the resistance phenotype. To confer resistance to antifolates, a mutant DHFR cDNA has to be introduced; by contrast, expression of only the wild-type MDR gene is sufficient to cause multidrug resistance. Gene-transfer studies to confer the MDR phenotype to the haemopoietic stem cells have shown that the transduced cells can exclude the panel of MDR drugs and thus become resistant. Clinical trials of retroviral vectors containing the MDR1 gene have been modestly successful at best. Although low gene expression was detectable for as long as a year after transplantation, the gene was truncated possibly owing to abnormal splicing. Improvements in gene-transfer methods (eg use of fibronectin, and the use of thrombopoietin in the cytokine cocktail) have allowed more efficient gene transfer. Although myelodysplasia has been observed in mice reconstituted with bone-marrow cells transduced with MDR1,42 this feature has not been seen in patients enrolled in clinical trials.39 An important enzyme that contributes to increased cellular resistance to nitrosoureas is MGMT. This enzyme prevents the generation of DNA interstrand cross-links induced by the chloroethylnitrosoureas by reacting with the cross-link precursors O6-chloroethylguanine or O6,N1ethanoguanine monoadducts in DNA and transferring the alkyl lesion to an internal cysteine residue.43 Moritz and colleagues44 reported that transduction of murine bonemarrow cells with a retroviral construct containing the MGMT gene confers resistance to carmustine. Infusion of MGMT-transduced bone-marrow progenitor cells to mice protected them from the toxic effects of weekly intraperitoneal injections of 40 mg/kg, strongly supporting the contention that gene transfer of MGMT may be useful for protection of the haemopoietic system from high-dose nitrosourea therapy. A recent development has been the generation of mutant forms of MGMT that are not sensitive to O6-benzylguanine, an inhibitor of this enzyme. Thus, transduced bone-marrow progenitor cells are protected from the toxicity of O6-benzylguanine and carmustine, whereas the tumour cells that contain the wild-type enzyme are sensitive.5,44 Table 1 summarises the various drugresistance genes that may be of use in myeloprotection and the chemotherapeutic agents against which resistance is conferred.28–31
Other gene products conferring drug resistance Genes involved in the antioxidant defence pathway, such as BCL2 and those encoding glutathione-associated enzymes, also confer drug resistance. The BCL2 (B-cell lymphoma/leukaemia 2) proto-oncogene was originally discovered because it is involved in t(14;18) translocations
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Conclusions Search strategy and selection criteria The literature search for this review was done on line with PubMed and the following search terms: ‘gene transfer’, ‘drug resistance’, and ‘myeloprotection’ from 1980 onwards for publications in English. Those provided as guides to further reading do not represent the entire literature on the subject and we apologise to the investigators whose contributions were not listed owing to space constraints. common to many non-Hodgkin lymphomas. It is an antiapoptotic protein. The t(14;18) translocations place the BCL2 gene at 18q21 in juxtaposition to the immunoglobulin heavy-chain locus at 14q32, resulting in transcriptional deregulation of BCL2. Induction of apoptosis by such external stimuli as radiation, hyperthermia, growth-factor withdrawal, glucocorticoids, and many classes of chemotherapeutic agents, is inhibited by BCL2 in vitro. Gene-transfer studies involving transfection of BCL2 cDNA into cells has unequivocally shown that cells transfected with, and overexpressing, this gene become resistant to various chemotherapeutic agents. Cells overexpressing BCL2 generate peroxides, but their cellular constituents including lipid membranes are not damaged. In mice, retrovirusmediated gene transfer of the BCL2 gene into bone-marrow cells protects the transplanted marrow cells from chemotherapy-induced myelosuppression without compromising the antitumour activity of the administered chemotherapeutic agent. Reconstitution of mice with these transduced marrow cells resulted in complete myeloprotection, compared with controls, when challenged with a myelosuppressive drug. Long-term adverse effects of BCL2 overexpression have not been studied,49 however, and such studies are needed. Glutathione-associated enzymes are important in cellular detoxification of xenobiotics. The glutathione-Stransferase (GST) enzyme family is particularly important with respect to anticancer-drug resistance. Four major isoforms of cytosolic GST have been described in mammalian cells. They share homology and are therefore thought to belong to the GST supergene family.50,51 The GST Π and the GST-Yc forms have been implicated in cellular resistance to nitrogen mustards and nitrosoureas, respectively. Transfection of the GST isoform cDNAs into breast-cancer cells (MCF7) did not produce convincing resistance to anticancer drugs. However, MCF7 cells have one of the lowest intrinsic GST-specific activities and thus have an inherently low capacity for synthesis of reduced glutathione (GSH). Therefore, in these cells, the GSH pathway may not be an important detoxification mechanism. Transfection of rat GST-Yc gene into rat mammary tumour cells and transfection of cDNAs of other GST enzymes into yeast and Chinese hamster ovary cells resulted in significantly higher resistance to anticancer drugs such as nitrogen mustards, cisplatin, and carboplatin. Gene transfer of the rat Yc gene into NIH 3T3 mouse fibroblasts and into the haemopoietic system in vitro resulted in an increase of five to ten fold in resistance to chlorambucil and mechlorethamine.51 THE LANCET Oncology Vol 3 March 2002
Cancer gene therapy has come a long way, from the euphoria of early unrealistic promises to the present-day realities. Obstacles such as poor efficiency of gene transfer, lack of tumour specificity, loss of sustained expression, and undesired toxic effects need to be addressed. Fresh hope comes from the recent success in retrovirus-mediated gene transfer and expression of the γC cytokine receptor subunit and subsequent correction of the severe combined immunodeficiency phenotype in two infants52 and correction of haemophilia due to deficiency of factor IX by transfer of the factor IX gene with adenoassociated virus.53 These successes are encouraging, but much remains to be done. As with any form of new treatment, cancer gene therapy, including myeloprotection strategies, has to undergo rigorous clinical trials of efficacy and long-term safety. Transfer and expression of drug-resistance genes in stem cells of cancer patients should theoretically permit safer dosing and dose-intensive chemotherapy. Future clinical trials should include poor-risk patients with largecell lymphoma, testicular cancer, and choriocarcinoma, who are likely to benefit from such an approach. Patients with small-cell lung cancer, or breast, ovarian, or bladder cancer may also benefit. Phase II trials should examine the possibility that there is an improved therapeutic index compared with historical controls, and phase III trials should compare post-transplant treatment (tolerance and efficacy) in patients transplanted with and without drugresistance genes. Acknowledgments
DB and JRB are supported by grants from the National Cancer Institute: CA-59350, CA-61586, and CA-86438. References
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