Joanna S. Remack

Tumor-Targeted Enzyme/Prodrug Therapy Mediates Long-term Disease-Free Survival of Mice Bearing Disseminated Neuroblastoma

Neural stem cells and progenitor cells migrate selectively to tumor loci in vivo. We exploited the tumor-tropic properties of HB1.F3.C1 cells, an immortalized cell line derived from human fetal telencephalon, to deliver the cDNA encoding a secreted form of rabbit carboxylesterase (rCE) to disseminated neuroblastoma tumors in mice. This enzyme activates the prodrug CPT-11 more efficiently than do human enzymes. Mice bearing multiple tumors were treated with rCE-expressing HB1.F3.C1 cells and schedules of administration of CPT-11 that produced levels of active drug (SN-38) tolerated by patients. Both HB1.F3.C1 cells and CPT-11 were given i.v. None of the untreated mice and 30% of mice that received only CPT-11 survived long term. In contrast, 90% of mice treated with rCE-expressing HB1.F3.C1 cells and 15 mg/kg CPT-11 survived for 1 year without detectable tumors. Plasma carboxylesterase activity and SN-38 levels in mice receiving both rCE-expressing HB1.F3.C1 cells (HB1.F3.C1/AdCMVrCE) and CPT-11 were comparable with those in mice receiving CPT-11 only. These data support the hypothesis that the antitumor effect of the described neural stem/progenitor cell-directed enzyme prodrug therapy (NDEPT) is mediated by production of high concentrations of active drug selectively at tumor sites, thereby maximizing the antitumor effect of CPT-11. NDEPT approaches merit further investigation as effective, targeted therapy for metastatic tumors. We propose that the described approach may have greatest use for eradicating minimum residual disease.

Development of a Tumor-Selective Approach to Treat Metastatic Cancer

Background Patients diagnosed with metastatic cancer have almost uniformly poor prognoses. The treatments available for patients with disseminated disease are usually not curative and have side effects that limit the therapy that can be given. A treatment that is selectively toxic to tumors would maximize the beneficial effects of therapy and minimize side effects, potentially enabling effective treatment to be administered. Methods and Findings We postulated that the tumor-tropic property of stem cells or progenitor cells could be exploited to selectively deliver a therapeutic gene to metastatic solid tumors, and that expression of an appropriate transgene at tumor loci might mediate cures of metastatic disease. To test this hypothesis, we injected HB1.F3.C1 cells transduced to express an enzyme that efficiently activates the anti-cancer prodrug CPT-11 intravenously into mice bearing disseminated neuroblastoma tumors. The HB1.F3.C1 cells migrated selectively to tumor sites regardless of the size or anatomical location of the tumors. Mice were then treated systemically with CPT-11, and the efficacy of treatment was monitored. Mice treated with the combination of HB1.F3.C1 cells expressing the CPT-11-activating enzyme and this prodrug produced tumor-free survival of 100% of the mice for >6 months (P<0.001 compared to control groups). Conclusions The novel and significant finding of this study is that it may be possible to exploit the tumor-tropic property of stem or progenitor cells to mediate effective, tumor-selective therapy for metastatic tumors, for which no tolerated curative treatments are currently available.

A specific CpG methylation pattern of the MGMT promoter region associated with reduced MGMT expression in primary colorectal cancers.

The enzyme O6‐methylguanine‐DNA methyltransferase (MGMT) protects cells from the cytotoxic and mutagenic effects of alkylating agents. Approximately 20% of tumor cell lines lack MGMT activity and are highly sensitive to alkylating agents. In established cancer cell lines, MGMT expression appears to be correlated with methylation of residues in both the promoter and the body of the gene. The effect of methylation of the MGMT promoter on gene expression and carcinogenesis in primary tumors is unknown. We investigated methylation of the MGMT promoter region in primary colorectal cancers and normal colonic mucosa. We used five methylation‐sensitive restriction enzymes (BssHII, SacII, EagI, NaeI, and SmaI) and Southern blot analysis to assess methylation in 46 cancers and 22 controls. Methylation of EagI and NaeI sites was seen in 12 tumors but in none of the 22 normal colorectal mucosa specimens. This difference was statistically significant (P < 0.01). Methylation‐sensitive single‐nucleotide primer extension analysis of four additional cytosine residues confirmed methylation of the promoter region in the tumors identified by EagI and NaeI digestions and served to further quantitate the extent of methylation. Western blot analysis of 21 tumors revealed statistically significant lower MGMT expression in the eight tumors with methylation of the EagI and NaeI sites and nt −128 than in the 13 tumors lacking the methylation pattern (P < 0.05). MGMT activity was lower in tumors with methylation than in tumors that were not methylated. The difference was not, however, statistically significant. We conclude that a subset of colorectal tumors is characterized by a specific methylation pattern in the MGMT promoter associated with reduced MGMT expression. Mol. Carcinog. 24:90–98, 1999. ? 1999 Wiley‐Liss, Inc.

In vitro methylation of the human O6-methylguanine-DNA methyltransferase promoter reduces transcription.

Approx. 20% of human tumor cell lines (termed Mer-) are deficient in the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT; E.C.2.1.1.63). Such cells possess the MGMT gene and promoter sequences but have virtually no mRNA or protein. Cytosine methylation of gene sequences has been proposed as a mechanism by which MGMT could be suppressed in Mer- cells; however, the experimental evidence does not uniformly support this idea. We therefore investigated the effect of in vitro methylation of the MGMT promoter in a reporter gene construct transfected into cultured human cells. DNA methylation by HpaII or HhaI methylases suppressed the activity of the promoter, although the effect was not absolute. The occurrence of partial intracellular demethylation of promoter sequences may account for the incomplete inhibition of transcription. A model that attempts to reconcile the opposing views on the role of cytosine methylation in MGMT gene expression is presented.

Evidence that O6-alkylguanine-DNA alkyltransferase becomes covalently bound to DNA containing 1,3-bis(2-chloroethyl)-1-nitrosourea-induced precursors of interstrand cross-links

The reaction of partially purified human O6-alkylguanine-DNA alkyltransferase with 1,3-bis(2-chloroethyl)-1-nitrosourea-treated DNA resulted in formation of a DNA-protein covalent complex. Complex formation required active alkyltransferase and brief treatment of DNA with the drug. DNA lost its capacity to form the complex once drug-induced DNA interstrand cross-links were completely formed. These results are consistent with a model in which the transferase catalyzes cleavage at O6-guanine and transfer of the alkyl moiety in a putative O6, N1-ethanoguanine intermediate of cross-link formation. DNA-protein complex formation presumably results when the transferase accepts the N1-ethanoguanine-DNA structure, analogous to its acceptance of simple alkyl groups.

ICAM-2 expression mediates a membrane-actin link, confers a nonmetastatic phenotype and reflects favorable tumor stage or histology in neuroblastoma

The actin cytoskeleton is a primary determinant of tumor cell motility and metastatic potential. Motility and metastasis are thought to be regulated, in large part, by the interaction of membrane proteins with cytoplasmic linker proteins and of these linker proteins, in turn, with actin. However, complete membrane-to-actin linkages have been difficult to identify. We used co-immunoprecipitation and competitive peptide assays to show that intercellular adhesion molecule-2 (ICAM-2)/α-actinin/actin may comprise such a linkage in neuroblastoma cells. ICAM-2 expression limited the motility of these cells and redistributed actin fibers in vitro, and suppressed development of disseminated tumors in an in vivo model of metastatic neuroblastoma. Consistent with these observations, immunohistochemical analysis demonstrated ICAM-2 expression in primary neuroblastoma tumors exhibiting features that are associated with limited metastatic disease and more favorable clinical outcome. In neuroblastoma cell lines, ICAM-2 expression did not affect AKT activation, tumorigenic potential or chemosensitivity, as has been reported for some types of transfected cells. The observed ICAM-2-mediated suppression of metastatic phenotype is a novel function for this protein, and the interaction of ICAM-2/α-actinin/actin represents the first complete membrane-linker protein-actin linkage to impact tumor cell motility in vitro and metastatic potential in an in vivo model. Current work focuses on identifying specific protein domains critical to the regulation of neuroblastoma cell motility and metastasis and on determining if these domains represent exploitable therapeutic targets.

The origin of O6-methylguanine-DNA methyltransferase in Chinese hamster overy cells transfected with human DNA

Transfection of Chinese hamster ovary (CHO) cells with human DNA has been shown in several laboratories to produce clones which stably express the DNA-repair protein, O6-methylguanine-DNA methyltransferase (MGMT), that is lacking in the parent cell lines (Mex- phenotype). We have investigated the genetic origin of the MGMT in a number of such MGMT-positive (Mex+) clones by using human MGMT cDNA and anti-human MGMT antibodies as probes. None of the five independently isolated Mex+ lines has human MGMT gene sequences. Immunoblot analysis confirmed the absence of the human protein in the extracts of these cells. The MGMT mRNA in the lines that express low levels of MGMT (0.6-1.4 x 10(4) molecules/cell) is of the same size (1.1 kb) as that present in hamster liver. One cell line, GC-1, with a much higher level of MGMT (4 x 10(4) molecules/cell) has two MGMT mRNAs, a major species of 1.3 kb and a minor species of 1.8 kb. It has also two MGMT polypeptides (32 and 28 kDa), both of which are larger than the 25 kDa MGMT present in hamster liver and other Mex+ transfectants. These results indicate that the MGMT in all Mex+ CHO cell clones is encoded by the endogenous gene. While spontaneous activation of the MGMT gene cannot be ruled out in the Mex+ cell clones, the intervention of human DNA sequences may be responsible for activation of the endogenous gene in the GC-1 line.

Development of an etoposide prodrug for dual prodrug-enzyme antitumor therapy

Enzyme-prodrug approaches to cancer therapy, theoretically, have the potential to mediate tumor-selective cytotoxicity. However, even if tumor-specific prodrug activation is achieved, enzyme-prodrug systems investigated thus far comprised a single enzyme and a specific prodrug. Although targeted, such systems constitute single-agent therapy, which may be ineffective and/or may promote development of drug resistance. Therefore, a goal of our laboratories was to design and characterize a novel dipiperidinyl derivative of etoposide [1,4′-dipiperidine-1′-carboxylate-etoposide (dp-VP16)] that would act as a prodrug. We envisioned that dp-VP16 would be converted to the active chemotherapeutic agent VP-16 by the same rabbit carboxylesterase (rCE) that we have previously shown to efficiently activate the prodrug irinotecan (CPT-11). This dp-VP16 prodrug might then be used in combination with CPT-11, with both drugs activated by a single enzyme. We evaluated the ability of pure rCE and two human carboxylesterases, hCE1 and hiCE (hCE2), to activate dp-VP16 in vitro, and in neuroblastoma cell lines designed to express/overexpress each enzyme. In SK-N-AS neuroblastoma cell transfectants, expression of rCE or hiCE decreased the IC50 of dp-VP16 as a single agent by 8.3- and 3.4-fold, respectively, in growth inhibition assays. Purified hCE1 did not metabolize dp-VP16 in vitro and did not affect its IC50 in intact cells. The combination indices of sequential exposure to CPT-11 followed by dp-VP16 ranged from ∼0.4 to 0.6, suggesting that this combination produced greater-than-additive cytotoxicity in neuroblastoma cells expressing rCE. These data provide proof-of-principle that enzyme-prodrug therapy approaches comprised of prodrugs with complementary mechanisms of cytotoxicity that are activated by a single enzyme can be developed. [Mol Cancer Ther 2006;5(6):1577–84]