OBATOCLAX and ABT-737 induce ER stress responses in human melanoma cells that limit induction of apoptosis.

Anti-apoptotic Bcl-2 family proteins, in particular, Mcl-1, are known to play a critical role in resistance of human melanoma cells to induction of apoptosis by endoplasmic reticulum stress and other agents. The present study examined whether the BH3 mimetics, Obatoclax and ABT-737, which inhibit multiple anti-apoptotic Bcl-2 family proteins, would overcome resistance to apoptosis. We report that both agents induced a strong unfolded protein response (UPR) and that RNAi knockdown of UPR signalling proteins ATF6, IRE1α and XBP-1 inhibited Mcl-1 upregulation and increased sensitivity to the agents. These results demonstrate that inhibition of anti-apoptotic Bcl-2 proteins by Obatoclax and ABT-737 appears to elicit a protective feedback response in melanoma cells, by upregulation of Mcl-1 via induction of the UPR. We also report that Obatoclax, but not ABT-737, strongly induces autophagy, which appears to play a role in determining melanoma sensitivity to the agents.

The BH3-mimetic ABT-737 sensitizes human melanoma cells to apoptosis induced by selective BRAF inhibitors but does not reverse acquired resistance.

Although the introduction of selective v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors has been a major advance in treatment of metastatic melanoma, approximately 50% of patients have limited responses including stabilization of disease or no response at all. This study aims to identify a novel means of overcoming resistance of melanoma to killing by BRAF inhibitors. We examined the influence of the BH3-mimetic ABT-737 on induction of apoptosis by the selective BRAF inhibitor PLX4720 in melanoma cells with or without BRAF V600E mutation. Included were cell lines established from four patients before and during treatment with selective BRAF inhibitors and 3D spheroids derived from these cell lines. Cell lines with no or low sensitivity to PLX4720 underwent synergistic increases and increased rates of apoptosis when combined with ABT-737. This degree of synergism was not seen in cell lines without BRAF V600E mutations. Apoptosis was mediated through the mitochondrial pathway and was due in part to upregulation of Bim as shown by inhibition of apoptosis following small interfering RNA knockdown of Bim. Similar effects were seen in cell lines established from patients prior to treatment but not in lines from patients clinically resistant to the selective BRAF inhibitors and in 3D spheroids derived from these cell lines. These results suggest that combination of selective BRAF inhibitors with ABT-737 or the related orally available compound ABT-263 may increase the degree and rate of responses in previously untreated patients with V600E melanoma but not in those with acquired resistance to these agents.

Human melanoma cells under endoplasmic reticulum stress are more susceptible to apoptosis induced by the BH3 mimetic obatoclax.

Past studies have shown that melanoma cells have largely adapted to endoplasmic reticulum (ER) stress, and this is associated with up-regulation of the antiapoptotic proteins Bcl-2 and Mcl-1. In this report, we show that the BH3 mimetic obatoclax potently overcomes resistance of melanoma cells to apoptosis induced by ER stress. Obatoclax, as a single agent at nanomolar concentrations, was relatively ineffective in the induction of apoptosis in melanoma cells, but treatment with obatoclax at these concentrations in combination with the ER stress inducer tunicamycin (TM) or thapsigargin markedly enhanced apoptotic cell death. This was primarily because of the inhibition of Mcl-1 by obatoclax, in that cotreatment with TM and another BH3 mimetic ABT737, which does not antagonize Mcl-1, caused only minimal increases in apoptosis. Moreover, overexpression of Mcl-1 inhibited apoptosis to greater degrees than overexpression of Bcl-2. In addition to direct inhibition of Mcl-1 by obatoclax, the combination of obatoclax and TM caused strong up-regulation of the BH3-only protein Noxa. Small RNA interference knockdown of Noxa partially inhibited apoptosis induced by cotreatment with obatoclax and TM. Similarly, knockdown of Bak also blocked induction of apoptosis by the compounds. The Mcl-1/Bak interaction seemed to be disrupted more efficiently in melanoma cells cotreated with obatoclax and TM. Taken together, these results identify obatoclax as a potent agent that overcomes resistance of melanoma cells to ER stress-induced apoptosis and seem to have important implications in the use of BH3 mimetics in the treatment of melanoma.