453. The Histone Deacetylase Inhibitor FK228 Can Increase Adenovirus Transgene Protein Expression in Human LOX IMVI Melanoma Xenografts

Abstract

Adenovirus gene therapy is frequently inefficient because of low levels of coxsackie adenovirus receptor (CAR) the primary adenovirus receptor. Previously, we showed that treatment of a diverse group of cancer cell lines with the histone deacetylase inhibitor FK228 (FR901228, depsipeptide), a drug in phase II clinical trials for the treatment of peripheral and cutaneous T-cell lymphoma, caused increases in CAR and |[alpha]|v-integrin RNA levels. Treatment of cells with 1 ng/ml FK228 prior to adenovirus infection was associated with a 5-10 fold increase in adenovirus transgene expression. Increases in transgene expression were not found in cultured normal cells from breast, liver or kidney following similar FK228 treatment. The effect of FK228 treatment was examined in athymic mice bearing advanced-stage subcutaneous LOX IMVI or UACC-62 human melanoma xenografts. The efficacy of treatment was evaluated by analyzing CAR and |[alpha]|v-integrin RNA and protein. Both types of xenografts (n=3) had increased CAR RNA levels as determined by semi-quantitative RT-PCR analysis 6 h following the last of three treatments with 3.6 mg/kg FK228. The LOX IMVI xenografts showed an 8.0-fold (|[plusmn]|0.9) and the UACC-62 xenografts showed a 5.1-fold (|[plusmn]|0.4) increase over untreated mice. The LOX IMVI system was chosen for further study because of greater response to FK228. Mice with LOX IMVI xenografts (n=6) treated with single 3.6 mg/kg FK228 dose and analyzed at 6 h following drug administration showed a 10.7-fold (|[plusmn]|4.9) increase in CAR levels while at 24 h there was a 13.6-fold (|[plusmn]|4.3) increase. By comparison, CAR levels in the livers, kidneys and lungs from the same animals remained unchanged. Xenografts from mice (n=6) treated with 3.6 mg/kg and analyzed by western blot analysis 6 h following drug treatment showed no increase in CAR protein levels, however, analysis at 24 h showed a 9.2-fold (|[plusmn]|4.8) increase in CAR protein. Little change in |[alpha]|v-integrin RNA or protein was observed under any conditions. Based on these results xenograft-bearing mice (n=10) treated with a single dose of 3.6 mg/kg FK288 were given an intra-tumor injection of adenovirus carrying a GFP transgene (2E+9 VP) 24 h following FK228 administration and the xenografts were analyzed 24 h following virus injection. Analysis of RNA from the entire xenografts showed a 7.2-fold (|[plusmn]|4.5) increase in expression from the adenovirus GFP transgene in the FK228 treated mice. Immunohistochemistry of formalin-fixed paraffin-embedded xenografts using an antibody to GFP showed higher levels of GFP protein expression diffusely spread throughout the xenografts in FK228 treated mice 18 h and 24 h following adenovirus injection. Thus, as previously observed in vitro, the FK228 induced increase in adenovirus transgene expression also occurs in vivo. These studies suggest that FK228 treatment can increase the efficiency of adenovirus gene therapy in vivo.