Combination epigenetic therapy has efficacy in patients with refractory advanced non-small cell lung cancer. xenografts even at doses well below those required LG 100268 to impact somatic solid tumors. Low dose guadecitabine also sensitized refractory EC cells to cisplatin antitumor activity was associated with activation of p53 and immune-related pathways and the antitumor effects of guadecitabine were dependent on p53, a gene rarely mutated in TGCTs. These preclinical findings suggest that guadecitabine alone or in combination with cisplatin is usually a promising strategy to treat refractory TGCT patients. DNA methylation is usually mediated primarily by DNMT3A and DNMT3B [16]. The nucleoside analogs 5-aza-deoxcytidine (referred to here as 5-aza) and 5-aza-cytidine are potent DNA methyltransferase inhibitors (DNMTIs) [17]. We showed that EC cells are highly sensitive to low concentrations of 5-aza [18]. This sensitivity appeared to partially dependent on high expression of the pluripotency-associated methyltransferase, DNMT3B [18]. In the current study, we evaluate the effects of the clinically and pharmacological optimized demethylating agent guadecitabine (SGI-110) on EC cells and in an animal model of LG 100268 cisplatin refractory nonseminoma testicular cancer [19]. EC-derived cisplatin resistant cells and tumors were highly sensitive to guadecitabine and guadecitabine was also able to sensitize cisplatin resistant tumors to cisplatin. Further, we demonstrate that these antitumor effects are highly associated with activation of p53, a gene rarely mutated in TGCTs. Interestingly, immune pathway genes were also LG 100268 induced in EC tumors by guadecitabine, suggesting that tumor immune activation could enhance antitumor activity in the clinic. Together our findings provide strong rationale for further development of guadecitabine as a novel therapy to treat patients with cisplatin-refractory testicular cancer. RESULTS Cisplatin sensitive and resistant EC cells are highly sensitive to low concentrations of guadecitabine in a DNMT3B-dependent manner We previously exhibited that a variety of TGCT-derived EC cells lines are highly sensitive to low nanomolar concentrations of 5-aza [18]. However, 5-aza and other DNA methylation inhibitors (DNMTIs) are subject to rapid degradation by hydrolytic cleavage and deamination necessitating chronic intravenous infusion [20]. In anticipation of potential clinical assessment of demethylation therapy for TGCTs we assessed the effects of the second generation demethylating agent guadecitabine that is not subject to the same metabolism as other DNMTIs and can be given subcutaneously with a longer effective half-life and a more extended exposure windows compared to 5-aza [19]. Cisplatin sensitive EC cells, NT2/D1 and cisplatin resistant NT2/D1-R1 cells were highly sensitive to guadecitabine with an IC-50 of 5 nM (Physique ?(Figure1A).1A). This is in contrast to the effects of guadecitabine on somatic solid tumor cells HCT116, U20S and MCF7 that were relatively insensitive to guadecitabine at concentrations as high at 1 M. Further, pretreatment of cisplatin resistant NT2/D1-R1 cells with low concentrations of guadecitabine resensitized the cells to cisplatin (Physique ?(Figure1B).1B). In this experiment Des cells pretreated with guadecitabine were allowed to recover before treating with cisplatin such that the cells had a comparable growth rate to cells not pretreated with guadecitabine. We have linked 5-aza hypersensitivity in EC cells to high levels of the DNA methyltransferase, DNMT3B and provided evidence to suggest that the relative insensitivity of somatic cancer cells to 5-aza is due to low DNA methyltransferase levels and activity [18]. The sensitivity of cisplatin sensitive and resistant EC cells to guadecitabine was highly dependent on DNMT3B as DNMT3B knockdown results in robust guadecitabine resistance in NT2/D1 and NT2/D1-R1 cells (Physique ?(Figure2).2). These data suggest that EC cells are exquisitely sensitive to the novel DNMTI guadecitabine, in part due to high levels of DNMT3B. Open in a separate window Physique 1 EC cells are highly sensitive to low concentrations of guadecitabine(A) Cisplatin sensitive EC cells, NT2/D1, and cisplatin resistant cells, NT2/D1-R1, but not HCT116 colon cancer cells, U2OS osteosarcoma cells, or MCF7 breast malignancy cells are sensitive to low concentrations of guadecitabine. Guadecitabine was added for 3 days to exponentially growing cultures. Viable cell growth and survival were measured. All data points are the average of biological triplicates. Error bars are standard deviation. * 0.01 comparing drug treatments to vehicle control in the same cell line. (B) Pretreatment with low concentrations of guadecitabine restores cisplatin sensitivity to cisplatin resistant EC cells. NT2/D1-R1 cells were pretreated with vehicle or 10 nM guadecitabine for 3 days before replating and a 48-hour recovery period followed by indicated cisplatin treatments for 6 hours. Cell viability was measured 3 days later. All data points are the average of biological triplicates. Error bars are standard deviation. * 0.01 comparing NTD1-R1 to NT2D1-R1 + guadecitabine. Experiments were repeated twice with comparable results. Open in a separate window.