Abstract 3499: Identification of a novel mechanism by which the BH3 mimetic obatoclax sensitizes non-Hodgkin's lymphoma tumor cells to TRAIL-mediated apoptosis

Abstract

Abstract The BH3 mimetic chemical antagonist obatoclax (GX15-070, Gemin X Pharmaceuticals) has been used in vitro and nowadays also in clinical trials for its potential therapeutic effects in both solid and lymph node malignancies. The effects of obatoclax treatment alone or in combination with cytotoxic drugs results in the reversal of tumor cell resistance. Studies by us and others have reported that inhibition of over expressed anti-apoptotic gene products such as Bcl-2 and Bcl-XL can reverse resistance to TRAIL. Obatoclax was shown to sensitize TRAIL-resistant solid tumors to TRAIL-apoptosis, inhibiting diverse anti-apoptotic molecules like Bcl-2, Bcl-XL and Mcl-1 through direct binding. We hypothesized that treatment of B-NHL cell lines with Obatoclax will result in tumor cell sensitization to TRAIL apoptosis by a different mechanism. B-NHL Ramos cell line were treated with different concentrations of Obatoclax (7-28 nM) and TRAIL (2.5-20 ng/ml) resulting in significant potentiation of apoptosis and synergy. Western analysis revealed that treatment with obatoclax resulted in significant inhibition of several members of the Bcl-2 family such as inhibition of Mcl-1, Bcl-XL, XIAP, and cIAP 1/2. In addition, obatoclax dissociated Bak from Mcl-1 as determined by immunoprecipitation. We found that, obatoclax inhibited the expression/activity of several members of the NF-κB pathway including phospho-p65, IKK2, and phosphor-IκB-α. The NF-κB activity inhibition by obatoclax was assessed by EMSA. We examined whether obatoclax-induced sensitization to TRAIL resulted from both signaling of DR4 and DR5. Treatment of Ramos cells with DR4 siRNA or DR5 siRNA and then treated with obatoclax and TRAIL resulted in the sensitization of cells treated with DR4 siRNA but not DR5 siRNA. We have previously reported that DR5 is negatively regulated by the transcription repressor YY1, upstream of NF-κB. We found that obatoclax inhibited YY1 transcription and expression. The direct role of YY1 inhibition by obatoclax in both DR5 upregulation and sensitization to TRAIL was determined in transfected cells with YY1 siRNA. Such cells showed upregulation of DR5 and sensitivity to TRAIL apoptosis. The inhibitory effect of obatoclax on Mcl-1 activity and the Mcl-1 direct role in sensitization was examined by treatment of cells with Mcl-1 siRNA. Such cells reversed their sensitivity to TRAIL-mediated apoptosis. Overall, the present findings reveal a new mechanism of obatoclax-induced sensitization to TRAIL by inhibiting NF-κB and downstream Mcl-1 and YY1. Both Mcl-1 and YY1 play a direct role in tumor cells sensitization to TRAIL mediated apoptosis. The present findings identify several new therapeutic targets modified by obatoclax whose modifications can reverse tumor cell resistance to TRAIL mediated apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3499. doi:10.1158/1538-7445.AM2011-3499