Is antisense nonsense?

In this issue of Leukemia and Lymphoma, Chanan-Khan et al. report a negative phase-3 study of oblimersen (an 18-mer-phosphorothioate oligonucleotide complimentary to the first six codons of the Bcl-2 open reading frame) and dexamethasone compared with dexamethasone alone in a heavily pre-treated group of patients with multiple myeloma [1]. Antisense oligonucleotides are short sequences of DNA that have been designed to hybridise to a specific target messenger RNA and can inhibit its translation into protein [2]. Antisense oligonucleotides are single-stranded DNA molecules usually 13–25 nucleotides long that hybridise to corresponding RNA and inhibit messenger RNA function and resultant inhibition of protein translation [3].

The B-cell leukemia gene 2 (Bcl-2) is one of the major apoptosis regulatory gene families. The apoptosis-suppressing Bcl-2 gene is a proto-oncogene located at the breakpoint of t(14;18) found in low-grade B-cell non-Hodgkin lymphomas, chronic lymphatic leukemia and Waldenström's macroglobulinemia [4]. Phase III trials in myeloma and AML did not meet their primary endpoints. The oncology drug advisory committee did not find substantial evidence of effectiveness in a metastatic melanoma phase III trial.

Several human Bcl-2 family proteins suppress apoptosis. Elevations in the levels of Bcl-2 are observed in several types of cancer. The Bcl-2 family proteins have been implicated in the resistance to cancer therapies which induce apoptotic cell death as their common basis [5]. Bcl-2 expression results in multi-drug resistance that prevents apoptosis induction by radiation and chemotherapy. Approaches to down-regulating the Bcl-2 protein by antisense therapy have been pursued as an anti-tumor strategy. Antisense oligonucleotides to Bcl-2 enter the cell expressing the gene, silencing the gene's protein product which should have an antiproliferative effect. The binding to the target messenger RNA should occur with high specificity and suppress the translation of the targeted message protein. If Bcl-2 is essential for survival of the B-cell, blocking its production should cause cell death.

The importance of the Bcl-2 target has been demonstrated in animal models. Transgenic mice expressing Bcl-2 accumulate an excess of mature B-lymphocytes. High concentrations of Bcl-2 are associated with relapse in AML and ALL. Bcl-2 blocks caspase activation in tumor cells preventing apoptosis induced by radiation and chemotherapy. Twenty-one patients with Bcl-2 positive relapsed non-Hodgkin lymphoma received a 14-day subcutaneous infusion of oblimersen [6]. No important systemic toxicity was seen in eight cohorts of patients. Dose-limiting toxicities have been thrombocytopenia, hypotension, fever and asthenia. Responses were seen, and documentation of reduced Bcl-2 activity was present in 7 of 16 patients. In a myeloma cell line, oblimersen does show activity. Bcl-2 protein is commonly expressed in multiple myeloma cell lines [7] and in vitro oblimersen sodium resulted in a time- and dose-dependent decrease in Bcl-2 RNA measured by RT-PCR. There was a greater than 75% reduction of Bcl-2 protein levels in the plasma cells after four days of exposure. A second in vitro study using myeloma cell lines and freshly isolated multiple myeloma cells demonstrated that the cytotoxic effect of dexamethasone and paclitaxel was affected by intracellular levels of Bcl-2. The use of antisense oligonucleotides over 1–4 days resulted in an increase in apoptosis at levels of 5 μg/mL and a 60% decrease in Bcl-2 protein levels after four days. When Bcl-2 antisense therapy was used on a Waldenström's macroglobulinemia cell line in culture, down-regulation of Bcl-2 with an increase in death of Waldenström's macroglobulinemia cells was seen.

Randomised phase 3 trials of oblimersen have been reported for four different indications: malignant melanoma, CLL, myeloma and AML. Phase 1 and 2 trials are also underway for a range of other cancers including prostate cancer. Oblimersen obtained orphan drug status in the United States and European Union for CLL in September 2001. A recently published phase 2 multicentre study of oblimersen in combination with rituximab in recurrent B-cell non-Hodgkin lymphoma demonstrated an overall response rate of 42% with 10 complete responses and 8 partial responses. Three of the responders were refractory to prior treatment with rituximab alone. The median duration of response was 12 months indicating that oblimersen sodium can be safely combined with rituximab and warrants further investigation [8]. Nonetheless, the effects of this agent apparent to be modest, although some of its off-target effects may represent useful therapeutic principles. Improvements in antisense cellular uptake may provide additional benefits. Further exploration of how targeted therapy molecules advance the therapy of cancer have as yet to be translated into improved outcomes for patients.