Enhancing Hemopoietic Drug Resistance: a Rationale for Reconsidering the Clinical Use of Mitozolomide
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© 2000 Nature America, Inc. 0929-1903/00/$15.00/ϩ0 www.nature.com/cgt Enhancing hemopoietic drug resistance: A rationale for reconsidering the clinical use of mitozolomide Leslie J. Fairbairn,1 Nachimuthu Chinnasamy,1,2 Linda S. Lashford,3 Dhanalakshmi Chinnasamy,2 and Joseph A. Rafferty1,2 Cancer Research Campaign Sections of 1Hemopoietic Cell and Gene Therapeutics and 2Genome Damage and Repair, Paterson Institute for Cancer Research, Manchester, United Kingdom; and 3Academic Department of Pediatric Oncology, Christie Hospital, National Health Service Trust, Manchester, United Kingdom. Retroviral gene transfer was used to achieve expression in mouse bone marrow of a mutant form of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA), which exhibits resistance to inactivation by O6-benzylguanine (O6-beG). After reconstitution of mice with transduced bone marrow, ϳ50% of the bipotent granulocyte-macrophage colony-forming cell (GM-CFC) and multipotent spleen colony-forming unit (CFU-S) hemopoietic populations showed expression of the transgene; this expression was associated with resistance to either mitozolomide or to a combination of O6-beG and mitozolomide, relative to mock-transduced controls. Thus, at a dose of mitozolomide in vivo that allowed only 70% and 62% survival of mock-transduced GM-CFC and CFU-S, respectively, the hATPA/GA CFC were totally resistant to the same dose of mitozolomide (P Ͻ .05 and .001, respectively). In the presence of O6-beG, the toxicity of mitozolomide was greatly potentiated. Only 24% and 18%, respectively, of mock-transduced GM-CFC and CFU-S survived combination treatment, whereas 45% (P Ͻ .05) and 37% (P Ͻ .01) of GM-CFC and CFU-S, respectively, from hATPA/GA mice survived the same combination of doses. Furthermore, as a result of trans- gene expression, the number of micronucleated polychromatic erythrocytes induced by mitozolomide was significantly reduced (P Ͻ .05) by 40% relative to mock-transduced controls, indicating the potential of this approach to reduce the frequency of mutation associated with chemotherapy exposure. The protection against the toxic and clastogenic effects of mitozolomide in both primitive and more mature hemopoietic cells suggests that the severe myelosuppression that halted further clinical investigation of this drug could be substantially ameliorated by the exogenous expression of O6-alkylguanine-DNA alkyltransferase. Therefore, these data raise the prospect for the reinvestigation of mitozolomide and other proscribed drugs in the context of genetically protected hemopoiesis. Cancer Gene Therapy (2000) 7, 233–239 Key words: Drug resistance; mitozolomide; alkylating agents; hemopoiesis; chemotherapy ne of the major drives in cancer therapy in the last toxicity becomes too severe, can result in limitation of O40 years has been the development of drugs that are chemotherapeutic dose and in inadequate tumor kill. capable of killing tumor cells. Many of these drugs are Often this problem of the inherent sensitivity of normal DNA-damaging agents that exert their effects via inter- cells is exacerbated by the development of drug resis- ference with the DNA replicative machinery, either tance in malignant cells, a situation which may leave directly or by the production of damage that prevents normal tissues more sensitive to cytotoxicity than the efficient cell division. As a result, tumor cells (which tumor. often have a higher proliferative index than surrounding These problems are exemplified by the O6-alkylating normal tissue) can prove sensitive to such agents. Un- agents. This family of drugs, which includes the nitro- fortunately, a number of normal tissues, particularly soureas and related methylating agents, are distin- those with a high proliferative potential, such as tissues guished by their ability to cause alkylation at the O6 of the lung, gut, and hemopoietic system, also exhibit position of guanine in DNA. In turn, O6-alkylguanine sensitivity to antitumoral agents. This leads to collateral (O6-alkG) appears to be the predominant cytotoxic as toxicity in patients undergoing chemotherapy and, if the well as mutagenic and carcinogenic lesion produced by these agents. The clinical use of O6-alkylating agents is Received February 8, 1999; accepted May 16, 1999. associated with acute toxicities in the bone marrow Address correspondence and reprint requests to Dr. Leslie J. Fairbairn, (BM), lung, and gut, with chronic lung toxicity, and with 1–4 Section of Hemopoietic Cell and Gene Therapeutics, Paterson Institute the induction of iatrogenic leukemias. for Cancer Research, Christie Hospital, National Health Service Trust, During its development in the mid 1980s, the imida- Manchester, United Kingdom M20 4BX. zotriazene mitozolomide showed significant antitumoral Cancer Gene Therapy, Vol 7, No 2, 2000: pp 233–239 233 234 FAIRBAIRN, CHINNASAMY, LASHFORD, ET AL: MITOZOLOMIDE AND ENHANCED HEMOPOIETIC DRUG RESISTANCE activity in preclinical murine models.5–7 After phase I zerland), recombinant murine IL-3 was obtained from R&D clinical trials, the primary dose-limiting activity was systems (Abingdon, UK), and recombinant rat stem cell factor determined to be thrombocytopenia, with evidence of was obtained from Amgen (Thousand Oaks, Calif). dose-related, but not severe, gut toxicity.8,9 However, on Producer cells progression to phase II trials, it became clear that although activity was seen in melanoma and small cell The LhATPA/GA vector that expresses the human carcinoma of the lung, the unpredictable and often hATPA/GA protein was constructed by cloning the hATPA/GA cDNA32 into the retroviral vector pLX derived severe myelosuppressive effects associated with treat- 36 ment were unacceptable.10–14 At this stage of develop- from the LN series of vectors. After derivation of ecotropic ment, mitozolomide was discontinued as a potential packaging cells producing LhATPA/GA vector, these cells were cocultured with amphotropic GP ϩ envAM12 cells in a antitumoral agent. “ping-pong” method to increase the viral titer. Amphotropic Resistance to O6-alkylating agents is largely mediated 6 producer cells were then selected by culturing in the presence by the DNA repair protein O -alkG-DNA alkyltrans- of 200 g/mL hygromycin for 7 days, and the resulting cultures 6 ferase (ATase), which transfers O -alkG to its active produced virus with a titer of in excess of 5 ϫ 105 infectious center in a stochiometric and autoinactivating man- particles/mL. ner.15–17 Expression of this protein in tumor cells corre- lates with their resistance to killing by O6-alkylating Animal studies 18,19 agents. Conversely, the extreme sensitivity of BM B6D2F1 male donor mice (9–12 weeks of age) were treated progenitors to such agents is a result of their low (often with 150 mg/kg 5-fluorouracil by intravenous injection. After 2 undetectable) levels of ATase.20 Attempts to improve days, BM cells were harvested from the femurs and cultured the therapeutic efficacy of the O6-alkylating agents have for 2 days at 37°C in Dulbecco’s modified Eagle’s medium centered on circumventing ATase-mediated tumor cell supplemented with 20% fetal calf sera, 0.1% bovine serum resistance. To this end, the use of analogs of O6-alkG, albumin, 2 mM glutamine, 10 ng/mL recombinant murine IL-3, such as O6-benzylguanine (O6-beG), has been demon- 100 ng/mL recombinant rat stem cell factor, and 200 U/mL recombinant human IL-6. The BM cells were scraped from the strated to lead to tumor sensitization if used in combi- ϩ 6 21–25 culture flasks and overlayed on either GP envAM12 (mock) nation with O -alkylating agent therapy. However, a or LhATPA/GA retroviral producer cells for 2 days in the major impediment of this strategy may be the lack of 6 same medium with the addition of 4 g/mL polybrene. Super- tumor specificity of O -beG, because a number of stud- natant cells were then collected, washed, and used either for ies have shown that this approach also leads to increased transplantation into 8- to 10-week-old, BM-ablated (15.2 Gy, 20,26–28 ϫ 6 killing of normal tissues. The discovery and de- Cobalt 60 source for 16 hours) female B6D2F1 mice (5 10 velopment of various mutant forms of ATase that exhibit cells per mouse) or for granulocyte-macrophage colony-form- different levels of resistance to inactivation by O6- ing cell (GM-CFC) colony plating in methylcellulose and beG29,30 has led to the possibility of developing a gene cytospin preparation. therapy strategy in which tumor sensitization might be To assess the gene transduction frequency among spleen colony-forming units (CFU-S), 10 recipient mice were trans- combined with normal tissue protection to increase the ϫ 6 31–35 planted with 1 10 cells each. The mice were sacrificed 12 therapeutic index. We have been investigating the days later and either spleens were fixed in 70% ethanol for utility of a double-mutated form of ATase (hATPA/GA) 6 6 immunocytochemical analysis or individual spleen colonies in providing O -beG-insensitive protection against O - were carefully dissected out for DNA isolation and subsequent 32–35 alkylating agent toxicity and clastogenicity. Here we polymerase chain reaction (PCR) analysis. report the effects of retroviral expression of this mutated At 1 month posttransplantation, groups of five mice were repair protein in hemopoietic cells on the in vivo biolog- treated with mitozolomide (1 mg/kg i.p.) either alone or 2 ical effects of mitozolomide and discuss the implications hours after receiving a single dose of O6-beG (30 mg/kg i.p). that the clinical use of this protective strategy, together After 24 hours, the treated mice were sacrificed by cervical dislocation, and femoral BM cells were harvested and used for with an otherwise proscribed drug, may have for cancer 28 treatment. CFU-S and GM-CFC analysis as described previously. PCR analysis MATERIALS AND METHODS PCR analyses were carried out on individual GM-CFC colo- nies picked from methylcellulose and on DNA isolated from day Materials 12 spleen colonies.