Enhancing Hemopoietic Drug Resistance: a Rationale for Reconsidering the Clinical Use of Mitozolomide

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 transgene 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 correlates 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 colonies picked from methylcellulose and on DNA isolated from day Materials 12 spleen colonies. A PCR product of 624 bp corresponding to the hAT cDNA was amplified by 30 rounds of PCR using the Mitozolomide was obtained from May-Baker (Dagenham, primers 5Ј-ATGGACAAGGATTGTGAAATGAAACG-3Ј and UK). It was dissolved in dimethylsulfoxide (100 mg/mL) and 5Ј-TCAGTTTCGGCCAGCAGGCGG-3Ј. Detection of male appropriately diluted in phosphate-buffered saline for intra- (donor) cells in the female recipients was performed by PCR peritoneal (i.p.) injection into mice. O6-beG was a generous using the primers 5Ј-AAGCGCCCCATGAATGCATT-3Ј and gift from Dr. R. S. McElhinney and Prof. B. McMurry (Trinity 5Ј-TCCCAGCTGCTTGCTGATCT-3Ј to amplify a 105-bp frag- College, Dublin, Ireland) and was homogeneously suspended ment of the murine Y chromosome-specific Sry gene.37 in corn oil using a Teflon glass homogenizer (Scientific Labo- ratory Supplies, Nottingham, UK) for i.p. injection into mice. Immunocytochemistry 5-fluorouracil (250 mg/mL solution) was purchased from David Bull Laboratories (Warwick, UK). Recombinant human Day 12 spleen colonies (3-␮m sections) and cytospin prepara- interleukin-6 (IL-6) was obtained from Sandoz (Basel, Swit- tions of transduced BM cells were stained with a rabbit

Cancer Gene Therapy, Vol 7, No 2, 2000 FAIRBAIRN, CHINNASAMY, LASHFORD, ET AL: MITOZOLOMIDE AND ENHANCED HEMOPOIETIC DRUG RESISTANCE 235

Figure 1. Immunohistochemical analysis of ATase expression in BM (A,B) and spleen colonies (C,D) of mock-transduced (B,D) and hATPA/GA-transduced (A,C) mice.

polyclonal antibody specific for human ATase using a strepta- RESULTS vidin biotin peroxidase complex detection system (Vector Laboratories, Peterborough, UK). Transduction of BM After in vitro transduction of murine BM with the Micronucleus test LhATPA/GA vector, the cells were harvested and used to reconstitute potentially lethally irradiated mice. Using Four control and seven LhATPA/GA-transduced mice were PCR analysis, it was established that 50% of BM GM- treated with a single dose of mitozolomide (0.5 mg/kg i.p) 2 6 CFC and CFU-S contained the transgene (data not hours after administration of O -beG (30 mg/kg i.p) in corn oil. shown). Immunohistochemical analysis of spleen colo- The treated mice were sacrificed 24 hours later, and femoral nies and BM showed that ATase was also expressed in marrow cells were flushed into fetal calf sera. BM smears were ϳ prepared from the cell pellets, fixed in methanol for 10 50% of these cells, in close agreement with the PCR minutes, and subsequently stained with acridine orange. The data. Furthermore, ATase protein was localized to the slides were scored independently by two separate investigators nucleus of transduced cells (Fig 1). for micronucleated polychromatic erythrocytes (MPCE) among 2000 polychromatic erythrocytes (PCE); results were Sensitivity of BM in vivo to mitozolomide- expressed as MPCE per 1000 PCE. induced toxicity To test the sensitivity of control and transduced BM to Statistical analysis killing by mitozolomide, mice were challenged with 6 The unpaired Student’s t test was used to compare the mitozolomide alone (1 mg/kg) or with O -beG (30 difference between experimental groups. mg/kg) 2 hours before administration of mitozolomide

Cancer Gene Therapy, Vol 7, No 2, 2000 236 FAIRBAIRN, CHINNASAMY, LASHFORD, ET AL: MITOZOLOMIDE AND ENHANCED HEMOPOIETIC DRUG RESISTANCE

Figure 2. In vivo survival of GM-CFC in control (filled bars) and hATPA/GA (open bars) mice after treatment with mitozolomide or mitozolomide plus O6-beG. Data shown are the mean Ϯ SEM of five P Ͻ .05, Student’s t test. Figure 4. Frequency of BM micronucleus induction in control (filled ,ء ;mice bars) and hATPA/GA (open bars) mice after treatment with mitozolo- .P Ͻ .05 ,ء ;mide and O6-beG. Data are the mean Ϯ SEM of five mice (1 mg/kg). Mice were sacrificed after 24 hours, and BM was taken for quantitation of GM-CFC (Fig 2) and compared with untransduced controls; 37% survival of CFU-S (Fig 3). Cells from control (untransduced) ani- CFU-S, P Ͻ .01 compared with untransduced mice). mals were extremely sensitive in vivo to the cytotoxic effects of mitozolomide. Thus, GM-CFC levels were Sensitivity of BM to mitozolomide- reduced to 70% of those in untreated mice by adminis- induced clastogenicity tration of mitozolomide alone, and to 24% of control levels by the combination of O6-beG and mitozolomide. To assess the effects of hATPA/GA transduction on the Similarly, CFU-S levels were reduced to 62% and 18% clastogenicity of mitozolomide in vivo, we used the of those in untreated mice after challenge with mitozo- mouse BM micronucleus assay (Fig 4). When BM from lomide or O6-beG/mitozolomide respectively. In con- either untransduced or hATPA/GA-transduced mice trast, however, the BM of mice that had been reconsti- was assessed in the absence of drug treatment, low levels tuted with hATPA/GA-transduced cells exhibited of MPCE were seen; these levels did not vary between substantial resistance to the cytotoxic effects of mitozo- the two groups, indicating that the transduction/trans- lomide. Thus, in the absence of O6-beG pretreatment, plantation protocol had no effect on the background no significant toxicity was seen in either the GM-CFC or micronucleus frequency. However, when untransduced 6 mice were exposed to mitozolomide (0.5 mg/kg) 2 hours CFU-S compartments. After pretreatment with O -beG, 6 both the GM-CFC and CFU-S populations exhibited a after pretreatment with O -beG, a 5-fold increase in the 2-fold increased survival over their counterparts from frequency of MPCE was observed (from 4 to 21 MPCE untransduced mice (44% survival of GM-CFC, P Ͻ .05 per 1000 PCE). In contrast, when hATPA/GA mice were treated with the O6-beG/mitozolomide combination, the frequency of micronucleus induction was significantly reduced compared with that seen in untransduced controls treated similarly (P ϭ .015).

DISCUSSION

In a number of preclinical studies, mitozolomide showed promising antitumoral activity.5–7 Phase I clinical trials identified a recommended oral dose of 115 mg/m2 for future investigations, with myelosuppression as the dose- limiting collateral toxicity.8,9 However, phase II trials quickly showed evidence of unpredictable and unacceptable myelotoxicity, particularly thrombocytopenia, with only marginal effects against tumors at the doses used 2 10–14 Figure 3. In vivo survival of CFU-S in control (filled bars) and (reduced to 90 mg/m to limit side effects). Given hATPA/GA (open bars) mice after treatment with mitozolomide or these effects, the use of mitozolomide in chemotherapy mitozolomide plus O6-beG. Data shown are the mean Ϯ SEM of five was discontinued. Aside from the myelosuppressive ef- P Ͻ .001, Student’s t test. fects of mitozolomide, one encouraging aspect of the ,ءءء ;P Ͻ .01 ,ءء ;mice

Cancer Gene Therapy, Vol 7, No 2, 2000 FAIRBAIRN, CHINNASAMY, LASHFORD, ET AL: MITOZOLOMIDE AND ENHANCED HEMOPOIETIC DRUG RESISTANCE 237 clinical trials was the lack of other serious side effects of protection against mitozolomide toxicity under circum- this agent. Indeed, in one trial in which BM support was stances in which substantial sensitization of tumors used to facilitate dose escalation, doses of up to 400 might be expected suggests that it may prove possible to mg/m2 were given, with the only significant toxicity being administer this agent in a therapeutically effective man- thrombocytopenia.38 Thus, it would seem that, given ner and dose, without undue myelosuppressive effects, proper protection of the hemopoietic compartment, it provided that the patient has been engrafted previously may prove possible to escalate mitozolomide doses to with autologous hATPA/GA-transduced hemopoietic clinically effective levels without undue adverse effects cells. Moreover, the reduced frequency of micronu- on other tissues. cleus (and by inference mutation) induction in the A number of genes have been identified that, when protected BM would imply that the risk of longer-term expressed in otherwise drug-sensitive tissues, can confer effects of mitozolomide treatment, such as secondary leu- protection against various cytotoxic agents; in addition, a kemias, will also be reduced. number of these genes have been expressed in BM39–42 Gene therapy clinical trials of ATase-mediated che- with a view toward providing chemoprotection in a moprotection will soon be undertaken. These will focus clinical context. The sensitivity of cells to the cytotoxicity initially on the potential for protection against agents and other biological effects of the O6-alkylating agents is such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) generally a function of the levels of the repair protein and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. Given ATase. In general, cells expressing high levels of ATase the effects of such drugs on tissues other than BM, it is are resistant to O6-alkylating agents,18,19,43–45 whereas unclear as to what window of opportunity for dose those (such as human hemopoietic cells) that have low escalation will be presented to the clinician before the levels are extremely sensitive.20,46 This situation is mag- next normal tissue toxicity leads to limitations in treat- nified by the fact that repair inactivates the ATase ment. For example, with BCNU, acute lung toxicity may protein; thus, there is a very strict correlation between be expected to present problems that will not be ame- the levels of ATase and drug sensitivity. Indeed, down- liorated by ATase expression in the BM.1,2 Moreover, no modulation of ATase levels via the use of O6-beG can be protection against the longer-term nonhemopoietic ef- used as a means of sensitizing otherwise resistant tumors fects (e.g., lung fibrosis) of such agents will be provid- to killing by O6-alkylating agents.21,23–25,47 However, the ed.3,4 One advantage in reconsidering mitozolomide is effects of O6-beG are not restricted to tumor tissue, and that this agent has not been associated with any signifi- the use of this inactivator before treatment with O6- cant nonhemopoietic toxicities in clinical trials, and so alkylating agents also results in toxicity to normal cells, may be particularly suited for use in conjunction with including those of the hemopoietic system.20,26–28 chemoprotective gene transfer. An additional advan- Thus, a strategy has emerged whereby it may prove tage, especially in relation to temozolomide, may lie in possible to enhance the therapeutic effects of the O6- the way chloroethylating agents such as mitozolomide alkylating agents; this strategy incorporates the use of kill cells. The mode of toxicity of this class of agent is potentially powerful tumor sensitizers such as O6-beG. likely to be via the generation of interstrand DNA Mutant forms of ATase have been characterized that cross-links produced by the reaction of the cyclic inter- exhibit resistance to inactivation by O6-beG while retain- mediate N-1-O6-ethanoguanine with the cytosine resi- ing the ability to repair O6-alkG in DNA.29,30 We have due of the opposite strand. As this would block DNA been working with a double-mutated form of human replication, it is likely to be fatal for the cell. The half-life ATase, hATPA/GA, and have shown previously that it of O6-chloroethylguanine adducts is ϳ6 hours; after this can confer O6-beG-insensitive protection against the time, they are not likely to be repaired by ATase. The cytotoxic effects of the methylating agent temozolomide, mechanism of cell killing by methylating agents such as one of the compounds that has replaced mitozolomide temozolomide is somewhat more obscure. One possibil- in the clinic.32–34 Here we show that retrovirus-mediated ity is that the O6-methylguanine (O6-meG):thymine mi- gene transfer and expression of hATPA/GA in mouse spair generated as a result of a round of DNA replica- BM leads to substantial, multilineage protection against tion may be a substrate for mismatch repair proteins. mitozolomide-induced cytotoxicity. Thus, after just a This may result in the cleavage of the newly synthesized single round of challenge with mitozolomide, a 2-fold strand and removal of the thymine residue. However, increased survival was seen in both the GM-CFC and during subsequent repair synthesis, the O6-meG adduct CFU-S populations. Furthermore, this protective effect will again miscode, and the mismatch repair substrate was maintained when mice were administered mitozolo- will be regenerated and recleaved, thereby resulting in mide in combination with the ATase inactivator O6-beG. an effectively permanent and potentially lethal strand Similarly, mice that were engrafted with hATPA/GA- break. More recently, it has also been shown that transduced BM showed a much reduced frequency of O6-meG can block DNA polymerase in vitro.48 If this micronucleus induction in the erythroid compartment occurs in vivo, it may also represent a major mode of compared with mock-transduced controls. Thus, we toxicity for these agents. However, both mechanisms of have demonstrated protective effects of hATPA/GA in methylating agent cytotoxicity require DNA replication; two mature compartments (granulocyte-macrophage thus, lesions may persist in DNA for perhaps up to 36 and erythroid) and one primitive compartment (CFU-S) hours before exerting any biological effect. Thus, the of the hemopoietic tissue. This multilineage in vivo toxicity associated with methylating agents may be com-

Cancer Gene Therapy, Vol 7, No 2, 2000 238 FAIRBAIRN, CHINNASAMY, LASHFORD, ET AL: MITOZOLOMIDE AND ENHANCED HEMOPOIETIC DRUG RESISTANCE promised somewhat by de novo resynthesis of ATase trial of mitozolomide in patients with advanced ovarian after depletion by O6-beG. The more rapid conversion cancer: a study of the EORTC Gynecological Cancer of O6-chloroethylguanine to a toxic intermediate may be Cooperative Group. Acta Oncol. 1989;28:663–665. less affected by the resynthesis rate. Certainly, this has 12. Smyth JF, Gundersen S, Renard J, Pinedo HM. Random- been the case for the chloroethylating agent BCNU, ized phase II trial of TCNU versus mitozolomide in malignant melanoma. EORTC Early Clinical Trials which has proved more toxic than temozolomide when 6 28 Group. Eur J Cancer Clin Oncol. 1989;25:755–757. administered to mice in combination with O -beG. If 13. Blackledge G, Roberts JT, Kaye S, et al. 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