Reactivation of the tumor suppressor RASSF1A by selective depletion of DNA methyltransferase 1 (DNMT1) by MG98 sensitizes renal cancer cells to interferon (IFN)-induced apoptosis

9541 Background: Epigenetic mechanisms may underlie resistance of renal cell carcinoma (RCC) to therapies including IFNs. RASSF1A is the tumor suppressor gene most frequently silenced by DNA methylation in renal cancer. Functions in mitosis control and apoptosis have been described. Methods: To inhibit maintenance of DNA methylation ACHN human renal cancer cells were treated with the DNMT1 selective phosphorothioate antisense inhibitor MG98 (DNMT1 AS) before exposure to IFNs. Flow cytometric TUNEL analysis and immunoblotting for caspase 3 cleavage were used to measure apoptosis. DNA demethylation was assessed by methylation specific PCR (MSP). Role of RASSF1A was examined by siRNA and lentiviral expression with selection of clones expressing physiologic levels, as determined by comparison on immunoblots to levels achieved by DNA demethylation. Results: Although ACHN cells were resistant to apoptosis induction by IFN-a2 or -β even at 500 U/ml (<5% TUNEL positive), MG98 (DNMT1 AS), but not mismatch oligonucleotide (DNMT1 MM) pretreatment led to apoptosis with only 50 U/ml IFN-a2 or -β (25–50% and 50–80% TUNEL positive, respectively). MG98 (DNMT1 AS) pretreatment alone did not result in apoptosis (<5% TUNEL positive) but decreased DNMT1 protein to undetectable levels, demethylated RASSF1A promoter region, and reactivated RASSF1A protein expression. RASSF1A siRNA transfection following demethylating treatment reduced IFN-induced apoptosis from 57% with control siRNA to 32% (TUNEL). Lentiviral stable expression of RASSF1A levels achievable by DNA demethylation overcame resistance of ACHN cells to IFN-induced apoptosis while empty virus did not sensitize (about 60% and 5% TUNEL positive, respectively, after 50 U/ml IFN-β). Untreated lentiviral clones had no increase in apoptosis (<5% TUNEL positive). Conclusions: DNA demethylation induced by MG98 treatment may enhance antitumor effect of IFNs in RCC at least in part by reactivation of RASSF1A. These results support the ongoing phase II clinical trial of IFN-a2 and MG98 in RCC. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration MethylGene MethylGene