Mechanism of Obatoclax (GX15-070; Bcl-2 Family Inhibitor)-Induced-Sensitization of B-NHL Cells to TRAIL-Mediated Apoptosis: Inhibition of the NF-κB Survival Pathway and the DR5 Transcription Repressor Yin Yang1 (YY1)

Patients with B-NHL respond initially to treatment with rituximab (chimeric anti- CD20 monoclonal antibody) in combination with CHOP. However, a subset of patients does not respond or develop refractoriness to further treatments. Therefore, there is an urgent need to develop novel therapeutic strategies to treat unresponsive patients. We have explored the potential therapeutic efficacy of TRAIL though, most tumors and cell lines are resistant to TRAIL-induced apoptosis. Our previous findings and those of others have demonstrated that the overexpression of anti-apoptotic gene products such as Bcl-2, Bcl_XL, and Mcl-1 regulates resistance to TRAIL and thus, inhibition of these gene products reverses resistance. Hence, we hypothesized that treatment of B-NHL cell lines with the Bcl-2 family inhibitor, Obatoclax (GX15-070; Gemin X Pharmaceuticals, Malvern, PA) will result in tumor cell sensitization to TRAIL apoptosis. We have used the B-NHL Ramos cell line as model. Treatment of Ramos cells with various concentrations of Obatoclax (7–28 nM) and TRAIL (2.5–20 ng/ml) resulted in significant potentiation of apoptosis and the combination treatment was synergistic. We then explored the mechanism of Obatoclax-induced sensitization to TRAIL. Treatment of Ramos cells with Obatoclax inhibited NF-κB activity and downstream anti-apoptotic gene products regulated by NF- κB (example Bcl-xl, Mcl-1 and XIAP) as assessed by western. Since Obatoclax inhibited NF-κB activity, we explored its effect on the transcription repressor YY1 and DR5 expression. Treatment of Ramos with Obatoclax significantly inhibited YY1 expression concomitantly with upregulation of total and surface DR5 expression that are regulated by NF-κB. The direct role of YY1 in the regulation of resistance to TRAIL was demonstrated by treatment of Ramos with siRNA YY1. Such treated cells showed upregulation of DR5 expression and sensitization to TRAIL apoptosis. The sensitization by Obatoclax resulted in activation of both Type I and Type II apoptotic pathways when used in combination with TRAIL. These findings establish a novel mechanism of Obatoclax-induced gene modification aside from its direct inhibition of Bcl-2 family. Further, our findings with Obatoclax are different from those recently reported by Song et al., [JBC 2008; July 3 (Epub ahead of print)] demonstrating that ABT-737, a small molecule Bcl-2 inhibitor, potentiated TRAIL-induced apoptosis via activation of NF-κB and NF-κB-induced upregulation of DR5 transcription via NF-κB DNA binding site on the DR5 promoter. It is possible that Obatoclax and ABT-737 mediate their sensitization to TRAIL via distinct mechanisms. In summary, our findings demonstrate the potential therapeutic application of Obatoclax in combination with TRAIL or agonist DR4/DR5 antibodies in the reversal of tumor cell resistance to TRAIL.