The cyclin-dependent kinase inhibitor PHA-848125 suppresses the in vitro growth of human melanomas sensitive or resistant to temozolomide, and shows synergistic effects in combination with this triazene compound.

PHA-848125 is a novel cyclin-dependent kinase inhibitor under Phase I/II clinical investigation. In this study, we describe, for the first time, the effect of PHA-848125 on human melanoma cells in vitro. Seven melanoma cell lines with different sensitivity to temozolomide (TMZ) were exposed to PHA-848125 for 5 days and then assayed for cell growth. In all cases, including TMZ-resistant cells, PHA-848125 IC(50) values were significantly below the maximum plasma concentrations achievable in the clinic. In the most PHA-848125-sensitive cell line, the drug caused a concentration-dependent G(1) arrest. PHA-848125 also impaired phosphorylation of the retinoblastoma protein at CDK2 and CDK4 specific sites, decreased retinoblastoma protein and cyclin A levels, and increased p21(Cip1), p27(Kip1) and p53 expression. Combined treatment with fixed ratios of TMZ plus PHA-848125 was studied in three melanoma cell lines. PHA-848125 was added to the cells 48 h after TMZ and cell growth was evaluated after 3 additional days of culture. Parallel experiments were performed in the presence of O(6)-benzylguanine (BG), to prevent repair of methyl adducts at O(6)-guanine induced by TMZ. Drug combination of TMZ plus BG and PHA-848125 produced additive or synergistic effects on cell growth, depending on the cell line. In the absence of BG, the combination was still more active than the single agents in the cell line moderately sensitive to TMZ, but comparable to PHA-848125 alone in the two TMZ-resistant cell lines. When TMZ plus BG were used in combination with PHA-848125 against cultured normal melanocytes, neither synergistic nor additive antiproliferative effects were observed. Our results indicate that PHA-848125 can have a therapeutic potential in melanoma patients, alone or combined with TMZ. Moreover this agent appears to be particularly attractive on the bases of its effectiveness against TMZ-resistant melanoma cells.

Novel role of triazenes in haematological malignancies: pilot study of Temozolomide, Lomeguatrib and IL-2 in the chemo-immunotherapy of acute leukaemia.

Previous studies indicated that dacarbazine and Temozolomide could be highly effective against refractory acute leukaemia. Their activity relies mainly on the generation of methyl adducts at the O(6)-position of guanine in DNA. High levels of O(6)-methylguanine-DNA methyltransferase (MGMT) or a defective mismatch repair (MMR) system, are associated with cellular resistance to triazenes. The MGMT inhibitor, O(6)-(4-bromothenyl)guanine (Lomeguatrib), can restore in vitro sensitivity to Temozolomide in MMR-proficient blasts. In the early 1970s we discovered that, in vivo, triazene compounds induce the appearance of novel transplantation antigens in murine leukaemia ("Chemical Xenogenization", CX). Non-self peptides presented by class I MHC molecules are generated by triazene-induced somatic mutations, affecting retroviral sequences that are detectable in the mouse genome. Moreover, preliminary experiments suggested that human cancer cells can also undergo CX. Therefore, we designed a chemo-immunotherapy strategy in leukaemic patients as follows: (a) cytoreduction and a hypothetical CX phase, i.e. treatment with Lomeguatrib (to suppress MGMT activity) and Temozolomide (to kill sensitive blasts and to presumably induce CX in resistant leukaemic cells); (b) immune response recovery phase using interleukin-2 (to possibly restore an immune response and take advantage of the hypothetical, triazene-induced CX). Here we present the results of pilot study which is in progress in patients with refractory/relapsed acute leukaemia. In all tested cases, Lomeguatrib suppressed MGMT activity in vivo. Six out of eight patients showed partial or complete disappearance of blast cells in peripheral blood or in bone marrow. We observed severe and long-lasting myelosuppression, accompanied by limited non-haematological toxicity. Up to now, two patients are alive (after 9 and 10 months, respectively), four died of opportunistic infections and two of progressive disease. This investigation confirms the potential role of triazenes in leukaemia and highlights the contribution of Lomeguatrib in overcoming drug resistance. Further studies are required to establish whether Temozolomide can induce CX in human leukaemia, and thus offer a new approach to control minimal residual disease.