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By Laszlo Dosa

WASHINGTON, DC - Designer peptides represent a new front in the war against uveal melanoma which could provide a more effective and less toxic treatment for the disease, reported US researchers at a Research to Prevent Blindness Foundation seminar.

Researchers at Washington University, St Louis, Missouri have identified several consistent molecular abnormalities in uveal melanoma and developed a peptide that triggers apoptosis in the malignant cells but not in normal cells.

This therapeutic peptide is effective against a wide range of eye cancer cells, both in culture and animal models.
The peptide targets three molecules. One of them, CDK2, is in the cell cycle pathway. Another is p53, a tumour suppressor which acts as a "surveillance checkpoint" and can initiate an apoptotic programme.

An important but surprising discovery about p53 has been that it is rarely mutated in uveal melanoma. This is surprising because it has been thought that cancer cells will undergo apoptosis unless p53 is inhibited or turned off.

"Uveal melanomas apparently block the function of p53 by over-expressing its major inhibitory protein, HDM2, which ensures tumour survival by preventing p53 triggering apoptosis.
"We hypothesised that a designer molecule which inhibits HDM2 may release p53 and allow it to trigger apoptosis in uveal melanoma cells," J. William Harbour MD explained.

The third molecular target for the designer peptide is the BCL2 pathway. Dr Harbour terms this the "commitment checkpoint" for apoptosis. Once this checkpoint is passed, the cell will irreversibly undergo apoptosis.
BCL2 is a family of pro-apoptotic and anti-apoptotic proteins that interact in a very complex manner and the net sum of that interaction will determine whether the apoptotic machinery will be activated.

"We developed peptides that inhibit the CDK2 and another that inhibits HDM2. That protein inhibits the key tumour suppressor protein, p53, which becomes activated during most abnormalities which develop in cancer formation. These include DNA damage and hyper-proliferative states where the cells divide too rapidly," Dr Harbour said.

Initially he tried to identify how these 'checkpoints' are abnormal in eye melanoma. He found that p53 was not mutated in most of these cancers but HDM2 was over- expressed in most.
The discovery led him to believe that this may be the way in which the cancer subverts p53 by over-expressing its inhibitor, thus keeping it in a functionally inactive state.

"We took human melanomas where the eye had to be enucleated and we have shown that over 90% of them over-express HDM2. Virtually all of them over expressed BCL2.
"Accordingly we felt these two abnormalities are consistently present in the ocular melanomas and so we decided to develop designer molecules that would inhibit either HDM2 or BCL2," he said.

In a novel approach, he created a competitive peptide that would specifically inhibit the interaction between HDM2 and p53.
That inhibition would allow the p53 to become activated. Similarly, he used another peptide that would inhibit BCL2 and allow pro-apoptotic proteins to become activated.

Early tests indicate some promising activity. The anti-HDM2 peptide, but not the control peptide, caused a rapid accumulation of activated p53 in cancer cells by blocking HDM2.
Within 24 hours, more than 80% of the cancer cells underwent apoptosis but the normal cells did not die, he reported.
Next, Dr Harbour tested the peptide against ocular cancer in rabbits. Human retinoblastomas were grown in the anterior chambers where they could be directly observed.

The anti-HDM2 peptide began to dissolve the tumours within 24 hours, whereas no effect was noted in the animals injected with a control peptide.
Three days later, microscopic examination of the eyes revealed that about 76% of the treated tumours were destroyed by apoptosis but the control peptide produced very little effect. Moreover, the anti-HDM2 peptide caused no detectable damage to normal ocular tissues.

Dr Harbour thinks the most likely way for his research to lead to a clinically relevant therapy would be to make the tumours more sensitive to radiation and/or chemotherapy because both procedures kill tumour cells by inducing apoptosis.

He believes that at least one of the reasons melanomas are resistant to radiation is that they have this defect which prevents the activation of p53. And there is also the BCL2 abnormality as well. Dr Harbour is now treating the cells with the designer peptides to see if he can make them more radiosensitive.

He pointed out that it is probably more realistic to believe the tumours could be made more sensitive to chemotherapy or radiation than to think the tumour could be eradicated altogether with the peptide.
"That would be the first goal - to use the peptide as an adjuvant to existing therapies.

And then maybe in the long term, it would evolve into something that would be the primary therapy for local treatment of the eye tumour or, potentially, something that we would deliver systemically to treat metastatic cancer.
"It would probably be in the next five years that we could potentially see the first trial as an adjuvant to radiotherapy," Dr Harbour said.

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