The BH3-Mimetic Obatoclax (GX15-070) Posses a Dual-Mechanism of Action and Induces Both Apoptosis and Autophagy-Dependent Cell Death of B Cell Non-Hodgkin’s Lymphoma (B-NHL) Cells

Abstract The Bcl-2 family of proteins represents a rheostat that controls cellular viability. Aberrant expression of these proteins is often found in B-cell non-NHL and is known to affect treatment outcomes in B-cell lymphoma patients. Novel compounds such as the BH3-mimetic Obatoclax have been designed to specifically target and counteract anti-apoptotic Bcl-2 family proteins. We previously demonstrated that Obatoclax was able to enhance the anti-tumor activity of rituximab and chemotherapy agents. In the current work, we studied the mechanisms by which Obatoclax induces its anti-tumor effects. Studies were conducted in rituximab-chemotherapy sensitive and resistant cell lines and in malignant B-cells derived from patients with B cell non-Hodgkin's lymphoma (n = 15). Cells were exposed to Obatoclax for 24 or 48 hrs with and without caspase inhibitors. Viability was evaluated using the cell-titer glow-assay. Induction of apoptosis was evaluated by flow cytometry and Western blotting. Autophagy was detected by LC3 conversion and electron microscopy. In addition, changes in p53, Noxa, Puma, and Bid following Obatoclax exposure were evaluated by Western blotting. Transient knock-down of p53 and beclin as well as pre-treatment of NHL cells to 3-methyladenine were performed to determine the role of of p53 or autophagy in the anti-tumor activity of Obatoclax, respectively. We found that Obatoclax induced time- and dose-dependent cell death of therapy-sensitive and –resistant cell lines along with all ex vivo treated patient samples tested to date (n = 15). In addition, in vitro exposure of NHL cells to Obatoclax resulted in significant downregulation of p53 and subsequent induction of Noxa and PUMA. Interestingly, the ability of Obatoclax to induce caspase-dependent PARP cleavage varied between patient samples and was not observed in therapy-resistant cell lines (TRCL). In Obatoclax rituximab-senstive treated cells, apoptosis was detected by APC-annexin staining. Inhibition of caspase activity by zVAD-fmk did not affect the ability of Obatoclax to kill B-NHL cell lines, suggesting that Obatoclax did not induce caspase-dependent apoptotic cell death of B-NHL cells. Induction of autophagy was detected by LC3 conversion not only in rituximab-chemotherapy sensitive or resistant cell lines but also in patient-derived tumor cells. Furthermore, electron microscopy confirmed the induction of autophagy following Obatoclax treatment of rituximab-chemotherapy resistant cells. Additionally, cell death induced by Obatoclax could be inhibited by knockdown of Beclin-1 or p53 and by treatment with 3-methyladenine. Together, these data suggest that Obatoclax has a dual mechanism of action and is capable to induce apoptosis or autophagy in B-cell NHL cells. We also provide data suggesting that both p53 and Beclin-1 have a pivotal role in response to Obatoclax and suggesting a crosstalk between p53, BH3 single domain proteins and autophagy. A better understanding in the molecular events triggered by Obatoclax and other Bcl-2 inhibitors is necessary in order to develop novel combination strategies using these exciting agents.