La Microempresa en Ecuador

The prognosis for patients with stage IV melanoma (spread beyond the regional lymph nodes) is very poor. Only 5% o f patients can be expected to survive 5 years (Koh 1991) as metastatic melanoma responds poorly to conventional treatments such as surgery, chemotherapy and radiotherapy.

Dacarbazine (DTIC) is the most effective chemotherapy drug used in metastatic melanoma patients, producing response rates of 10-20%. However, these responses are generally of short duration only, with long-term complete responses only seen in 1-2% o f patients (Hill et al. 1984). In addition the impact on visceral compared with skin and subcutaneous métastasés is poor. A number o f combination chemotherapy regimens have also been tested but few have shown advantages over DTIC alone (Legha et al. 1989, Lee et al. 1995), ( Margolin et al. 1998). The use o f interferon and

interleukin-2 in combination with multiple chemotherapeutic drugs (biochemotherapy) has been demonstrated to produce overall response rates of 62% with long-term durable complete remissions observed in up to 10% of patients (Legha et al. 1997).

There is therefore a clear need to develop more effective forms of therapy for patients with metastatic melanoma. This project has focussed on the use of corrective gene therapy in melanoma. Previous fellows have demonstrated the efficacy of antisense ODNs targeting the c-myc oncogene at inhibiting the growth o f melanoma in-vitro and in-vivo (Rajiv Grover MD thesis 1996, Jagdeep Ghana MD thesis 1998). This gene was targeted due to its strong prognostic significance in melanoma and therefore its likely important role in the development and progression of melanoma in patients. As the next stage in this project, the use of c-myc antisense in combination with other chemotherapeutic drugs was investigated in-vitro to assess if additive effects could be produced which might be o f benefit to patients with metastatic disease.

The combination o f c-myc antisense with cis-platinum, DTIC, Temozolomide and Taxol was investigated in the A375m human melanoma cell line. A large additive effect was demonstrated when c-myc antisense was combined with cis-platinum in the A375m melanoma cell line, which is known to over-express the c-myc oncogene. Calculation of the window o f theoretical additivity showed that the actual growth inhibitory effect produced by the combination was greater than would be expected by the addition o f the effects produced by the two agents acting individually. This suggests a synergistic mechanism.

In addition to the direct promotion of tumour growth, c-myc overexpression is known to alter the behaviour o f tumour cells in other ways which promote disease progression in melanoma, and other cancers (Evan et al. 1993). Overexpression o f c- myc down-regulates surface MHC molecule expression, thus reducing the anti- tumour immune response (Grover et al. 1996) and is associated with resistance to a number o f chemotherapy drugs, including cis-platinum (Kashani-Sabet et al. 1990, Sklar et al. 1991, Walker et al. 1996). This is the likely reason to explain the super­ additive effects observed in this experiment. Exposure of the A375m cells to c-myc antisense may have reversed a degree of cis-platinum resistance, therefore resulting in a much larger growth-inhibitory effect when the cells were subsequently treated with this drug.

The use of corrective gene therapy to block abnormal oncogene expression may therefore not only lead to inhibition of tumour growth, but may also increase tumour sensitivity to chemotherapeutic agents such as cis-platinum. This may offer important benefits to patients with metastatic melanoma in the future.

The other interesting result from this study was the potent effect o f Taxol against the A375m melanoma cell line. This highlights the potential efficacy o f this drug for the treatment o f metastatic melanoma patients. At present, although Taxol has demonstrated good efficacy against metastatic disease in ovarian and beast cancer patients, the response rates in metastatic melanoma have been relatively disappointing. The response rates produced by Taxol have been no better than those for DTIC (Aamdal et al. 1994). Taxol is thought to exert its anti-tumour effects in part by phosphorylation of the bcl-2 oncogene, thereby blocking its inhibitory action on apoptosis. However the bcl-2 gene is overexpressed in metastatic melanoma and its expression level has been shown to be an accurate prognostic marker in this condition (Grover et al. 1996). The efficacy o f Taxol has been shown to be greatly increased by overexpression of the bcl-Xsgene, which is an inhibitor of bcl-2 activity (Sumantran et al. 1995). Therefore, inhibition o f bcl-2 expression by other methods, such as by the use o f antisense, may potentially increase the efficacy o f Taxol. Combination o f Taxol and bcl-2 antisense therapy may therefore offer potential benefits for the treatment of metastatic melanoma.

In summary, the results in this chapter, along with those published by other groups (Mizutani et al. 1994, Nieborowska Skorska et al. 1994, Jansen et al. 1998), suggest that it is possible to increase the sensitivity of tumours to chemotherapeutic agents using corrective gene therapy. This offers obvious benefits to patients with advanced malignancy as their tumours become resistant to standard chemotherapeutic drugs, shortening their survival. The use o f corrective gene therapy to reverse chemoresistance, in combination with chemotherapeutic drugs, may therefore lead to the more effective treatment of metastatic disease in the future.

7.5

The development of a ribozyme targeting

the c-myc oncogene in melanoma

The demonstration that antisense ODN’s ean specifieally bloek the expression o f a target gene has highlighted this meehanism as a potential form o f eorrective gene therapy for use in melanoma, and a wide range o f other diseases. However, there are limitations whieh prevent the effeetive use of antisense ODNs in patients. Effective tumour-targeting is required and adequate expression levels must be achieved at the target site, for a sufficient period of time, to effectively block expression of the target gene and produce therapeutic effect.

As a first stage in the development of e-mye antisense for use in melanoma patients a hammerhead ribozyme targeting the e-mye oncogene was designed, synthesised and its efficacy assessed. Ribozymes are naturally-oeeuring forms o f RNA that possess the ability to cleave other RNA sequences by acting in an enzymic manner. Alteration o f the ribozyme structure to target specific RNA sequences produces a molecule whieh ean act in the same manner as antisense but is much more efficient. One molecule of antisense can only bloek the effects of one molecule of mRNA before being consumed in the process. However one ribozyme molecule targeting a specific RNA sequence ean cleave multiple RNA molecules.

A ribozyme was designed and synthesized, according to standard principles, to target the same translation-initiation codon o f exon 2 of e-mye mRNA whieh has been targeted by the 15-mer e-mye ODN used by previous RAFT fellows and other groups (Leonetti et al. 1996)(base pairs 296-311). Its efficacy in-vitro was subsequently tested in the A375m melanoma cell line.

Although transfection of the A375m cell line with the pREVmycl 7 ribozyme was shown to significantly reduce cell growth rates over a 6-day period, this effect was also shown by the pREV plasmid alone. This therefore implied that the effect was non-speeifie and most likely due to a transfeetion-related effect reducing the cellular growth rate.

This effect was confirmed to be non-speeifie by the demonstration that transfection o f A375m cells by the pREVmycl 7 ribozyme did not result in significant down- regulation o f e-mye gene expression.

There are a number of reasons why the pREVmycl? ribozyme may not have been effective against the c-myc oncogene. The ribozyme targeted a -GUU- sequence in exon 2 o f the c-myc mRNA. Although this sequence is known to be an effective target for ribozymes, the -GUC- sequence is known to be the best triplet to achieve the highest ribozyme cleavage efficiency. Alternatively, although the ribozyme may have had the primary nucleotide sequence to potentially bind to the translation- initiation site in exon 2, the 3-dimensional structure may have prevented the target nucleotide sequence from binding to the cleavage site.

There are therefore a number of different options available to produce an effective ribozyme which targets c-myc mRNA. Analysis of the 3-dimensional structure o f the pREVmycl? ribozyme may reveal if access to the cleavage site is blocked by the tertiary structure o f the ribozyme and aid the planning of a revised ribozyme structure targeting the same c-myc mRNA sequence.

Alternatively, a different base sequence o f the c-myc mRNA could be targeted, containing the triplet sequence -GUC-. Ohta et al demonstrated the successful use of three ribozymes targeting the ras, fos and c-myc oncogenes in a melanoma cell line transfected with the ras oncogene (Ohta et al. 1996). Their conclusions were that the ras ribozyme was the most effective as it reduced the cell growth rate the most. However, the ribozymes had been tested in a ras over-expressing cell line so this observation was not unexpected. Although there is likely to have been some secondary increase in c-myc expression in this cell line due to the increased replication rate, the c-myc level is likely to have been much lower than that o f the A3?5m cell line, which is a high c-myc expressor. Had the c-myc ribozyme been tested in a melanoma cell line expressing high levels o f c-myc its effects may have been greater and the conclusions might therefore have been different.

C-myc expression has been shown to be a highly significant prognostic factor for a number o f different types of primary and also metastatic melanoma (Grover et al. 199?, Grover et al. 199?, Ghana et al. 1998, Ross et al. 1998). This has not been demonstrated for either the ras or the fos oncogenes. Therefore, it is likely that the c- myc oncogene is a more useful target for corrective gene therapy in metastatic melanoma patients than either ras or fos, and the successful demonstration o f the efficacy of a ribozyme targeting the c-myc oncogene by Ohta et al is therefore important. The testing o f this ribozyme in a high c-myc-expressing melanoma cell